103P2D6: tissue specific protein highly expressed in various cancers

ABSTRACT

A novel gene (designated 103P2D6) and its encoded protein are described. 103P2D6 is not expressed in normal adult tissue, but is highly expressed in prostate tissue xenografts, providing evidence that it is turned on in prostate cancer. 103P2D6 is also expressed in some fetal tissues, and in breast, bladder, lung, bone, colon, pancreatic, testicular, cervical and ovarian cancers. Consequently, 103P2D6 provides a diagnostic and/or therapeutic target for cancers, and the 103P2D6 gene or fragment thereof, or its encoded protein or a fragment thereof can be used to elicit an immune response.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional application No. 60/184,558, filed Feb. 24, 2000, and U.S. provisional application No. 60/218,856, filed Jul. 13, 2000, the entire contents of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention described herein relates to a novel gene and its encoded protein, termed 103P2D6, and to diagnostic and therapeutic methods and compositions useful in the management of various cancers that express 103P2D6.

BACKGROUND OF THE INVENTION

[0003] Cancer is the second leading cause of human death next to coronary disease. Worldwide, millions of people die from cancer every year. In the United States alone, cancer causes the death of well over a half-million people annually, with some 1.4 million new cases diagnosed per year. While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. In the early part of the next century, cancer is predicted to become the leading cause of death.

[0004] Worldwide, several cancers stand out as the leading killers. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the primary causes of cancer death. These and virtually all other carcinomas share a common lethal feature. With very few exceptions, metastatic disease from a carcinoma is fatal. Moreover, even for those cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical debilitations following treatment. Furthermore, many cancer patients experience a recurrence.

[0005] Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is by far the most common cancer in males and is the second leading cause of cancer death in men. In the United States alone, well over 40,000 men die annually of this disease—second only to lung cancer. Despite the magnitude of these figures, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration and chemotherapy continue to be the main treatment modalities. Unfortunately, these treatments are ineffective for many and are often associated with undesirable consequences.

[0006] On the diagnostic front, the lack of a prostate tumor marker that can accurately detect early-stage, localized tumors remains a significant limitation in the diagnosis and management of this disease. Although the serum prostate specific antigen (PSA) assay has been a very useful tool, however its specificity and general utility is widely regarded as lacking in several important respects.

[0007] Progress in identifying additional specific markers for prostate cancer has been improved by the generation of prostate cancer xenografts that can recapitulate different stages of the disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immune deficient (SCID) mice and have exhibited the capacity to mimic the transition from androgen dependence to androgen independence (Klein et al., 1997, Nat. Med.3:402). More recently identified prostate cancer markers include PCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res September 1996;2(9):1445-51), STEAP (Proc Natl Acad Sci USA. Dec. 7, 1999;96(25):14523-8) and prostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

[0008] While previously identified markers such as PSA, PSM, PCTA and PSCA have facilitated efforts to diagnose and treat prostate cancer, there is need for the identification of additional markers and therapeutic targets for prostate and related cancers in order to further improve diagnosis and therapy.

SUMMARY OF THE INVENTION

[0009] The present invention relates to a novel gene, designated 103P2D6 that is over-expressed in multiple cancers listed in Table I. Northern blot expression analysis of 103P2D6 gene expression in normal tissues shows a restricted expression pattern in adult tissues. Analysis of 103P2D6 expression in normal prostate and prostate tumor xenografts shows over-expression in LAPC-4 and LAPC-9 prostate tumor xenografts. The nucleotide (FIG. 2) and amino acid (FIG. 2 and FIG. 3) sequences of 103P2D6 are provided. Portions of the 103P2D6 amino acid sequence show some homologies to ESTs in the dbEST database. The tissue-related profile of 103P2D6 in normal adult tissues, combined with the over-expression observed in prostate and other tumors, shows that 103P2D6 is aberrantly over-expressed in at least some cancers, and thus serves as a useful diagnostic and/or therapeutic target for cancers of the tissues listed in Table I.

[0010] The invention provides polynucleotides corresponding or complementary to all or part of the 103P2D6 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 103P2D6-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids; as well as the peptides/proteins themselves; DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides or oligonucleotides complementary or having at least a 90% homology to the 103P2D6 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 103P2D6 genes, mRNAs, or to 103P2D6-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 103P2D6. Recombinant DNA molecules containing 103P2D6 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 103P2D6 gene products are also provided. The invention further provides antibodies that bind to 103P2D6 proteins and polypeptide fragments thereof, including polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable marker.

[0011] The invention further provides methods for detecting the presence and status of 103P2D6 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 103P2D6. A typical embodiment of this invention provides methods for monitoring 103P2D6 gene products in a tissue or hematology sample having or suspected of having some form of growth dysregulation such as cancer.

[0012] The invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 103P2D6 such as prostate cancers, including therapies aimed at inhibiting the transcription, translation, processing or function of 103P2D6 as well as cancer vaccines.

BRIEF DESCRIPTION OF THE FIGURES

[0013]FIG. 1. shows the 103P2D6 suppression subtractive hybridization (SSH) DNA sequence (SEQ ID NO: 3).

[0014] FIGS. 2A-B. shows the nucleotide and amino acid sequences of 103P2D6. cDNA and ORF for 103P2D6 clone B, Kozak sequence and start methionine are indicated in bold. See Example 2, infra.

[0015]FIG. 3. shows the amino acid sequence encoded by the open reading frame shown in FIG. 2, and lists the amino acid positions used for proteins/peptides of the invention. The 103P2D6 signal sequence is boxed.

[0016]FIG. 4. shows the sequence alignment of 103P2D6 (ORF from clone B) with env protein from human endogenous retroviral HERV-H (FASTA accession: Q9UNM3). The 103P2D6 protein sequence has homology to the HERV-H env protein (24.9% identity and 32.8% homology taking account of any gaps).

[0017] FIGS. 5A-C. shows the northern blot analysis of 103P2D6 expression in various normal human tissues (using the 103P2D6 SSH fragment as a probe) and LAPC xenografts. Two multiple tissue northern blots (Clontech) and a xenograft northern blot were probed with the 103P2D6 SSH fragment. Size standards in kilobases (kb) are indicated on the side. Each lane contains 2 μg of mRNA for the normal tissues and 10 μg of total RNA for the xenograft tissues. The results show the expression of 103P2D6 in LAPC xenografts, and not in normal prostate and other tissues. Lanes in FIG. 5A represent (1) heart; (2) brain; (3) placenta; (4) lung; (5) liver; (6) skeletal muscle; (7) kidney; (8) pancreas. Lanes in FIG. 5B represent (1) spleen; (2) thymus; (3) prostate; (4) testis; (5) ovary; (6) small intestine; (7) colon; (8) leukocytes. Lanes in FIG. 5C represent (1) prostate; (2) LAPC-4 AD; (3) LAPC-4 Al; (4) LAPC-9 AD; (5) LAPC-9 AI.

[0018]FIG. 6 shows the northern blot analysis of 103P2D6 expression in various cancer cell lines. Lanes represent (1) LAPC-4 AD; (2) LAPC-4 Al; (3) LAPC-9 AD; (4) LAPC-9 Al; (5) LNCaP; (6) PC-3; (7) DU145; (8) TsuPr1; (9) LAPC-4 CL; (10) HT1197; (11) SCaBER; (12) UM-UC-3; (13) TCCSUP; (14) J82; (15) 5637; (16) 293T; (17) RD-ES; (18) PANC-1; (19) BxPC-3; (20) HPAC; (21) Capan-1; (22) SK-CO-1; (23) CaCo-2; (24) LoVo; (25) T84; (26) Colo-205; (27) KCL 22; (28) PFSK-1; (29) T98G; (30) SK-ES-1; (31) HOS; (32) U2-OS; (33) RD-ES; (34) CALU-1; (35) A427; (36) NCI-H82; (37) NCI-H146; (38) 769-P; (39) A498; (40) CAKI-1; (41) SW839; (42) BT20; (43) CAMA-1; (44) DU4475; (45) MCF-7; (46) MDA-MB-435s; (47) NTERRA-2; (48) NCCIT; (49) TERA-1; (50) TERA-2; (51) A431; (52) HeLa; (53) OV-1063; (54) PA-1; (55) SW626; (56) CAOV-3.

[0019]FIG. 7. shows the northern blot analysis of 103P2D6 expression in various LAPC-4 AD xenografts, including subcutaneously grown xenografts (sc), intratibially grown xenografts (it), and xenografts grown within human bone explants (LAPC-4 AD²) in SCID mice. Lanes represent (1) LAPC-4 AD sc; (2) LAPC-4 AD sc; (3) LAPC-4 AD sc; (4) LAPC-4 AD it; (5) LAPC-4 AD it; (6) LAPC-4 AD it; (7) LAPC-4 AD².

[0020]FIG. 8. shows a northern blot analysis of 103P2D6 expression in 9 week old fetal tissues, including 6 organs and whole embryo. Lanes represent (1) brain; (2) heart; (3) kidney; (4) liver; (5) lung; (6) muscle; (7) whole embryo.

[0021]FIG. 9 shows a RT-PCR Expression analysis of 103P2D6. cDNAs generated using pools of tissues from multiple normal and cancer tissues were normalized using beta-actin primers and used to study the expression of 103P2D6. Aliquots of the RT-PCR mix after 26 (upper portion of this figure) and 30 cycles (lower portion of this figure) were run on the agarose gel to allow semi-quantitative evaluation of the levels of expression between samples. The first strand cDNAs in the various lanes of this figure are as follows: Lane 1 (VP-1) contains liver, lung, and kidney first strand cDNA from normal tissues; lane 2 (VP-2) stomach, spleen, and pancreas from normal tissues; lane 3 (xenograft tissue pool) LAPC4AD, LAPC4AI, LAPC9AD, and LAPC9AI; lane 4 is normal prostate tissue pool; lane 5 is prostate cancer tissue pool; lane 6 is bladder cancer tissue pool; lane 7 is kidney cancer tissue pool; lane 8 is colon cancer tissue pool; lane 9 is from a lung cancer patient; and lane 10 is water blank.

[0022]FIG. 10. shows the results of RT-PCR analysis of 103P2D6 expression in patient-derived cancers. Lane 1 contains a sample from normal prostate; lane 2 from normal kidney; lane 3 from a prostate tumor pool; lane 4 from a kidney tumor pool; lane 5 from a bladder tumor pool; lane 6 from HeLa cells; and for lane 7 water was used.

[0023] FIGS. 11A-C. shows expression of 103P2D6 in pancreatic, colon, and prostate cancer cell lines. In panels A and B, cell lysates (˜25 μg) from the indicated cell lines were separated by SDS-PAGE and subjected to Western blot analysis using an anti-103P2D6 pAb. Indicated with an arrow is a strong anti-103P2D6 pAb itraunoreactive band of approximately 60 kD present in the pancreatic cancer cell lines HPAC and Bx PC-3, the colon cancer cell line CaCo-2, and a less intense band in LAPC9 prostate cancer cells indicative of endogenous 103P2D6 protein expression. Also indicated with an arrow is the 85 kD immunoreactive band present in 293T cells transfected with V5-His tagged 103P2D6 cDNA. In panel C, Bx PC-3 pancreatic cancer cells were stained with anti-103P2D6 pAb (10 μg/ml) or control rabbit IgG Ab and subjected to flow cytometric analysis following incubation with anti-rabbit IgG-FITC conjugated secondary Ab. Bx PC-3 cells stained with the anti-103P2D6 pAb exhibited a fluorescence shift compared to the cells stained with control rabbit IgG, indicating cell surface expression of 103P2D6.

[0024] FIGS. 12A-B shows expression of 103P2D6 protein in 293T cells. For the data in panel A, 293T cells were transiently transfected with either pcDNA 3.1 V5-His 103P2D6 plasmid or with empty control vector and harvested 2 days later. Cells were lysed in SDS-PAGE sample buffer and lysates were separated by SDS-PAGE gel and transferred to nitrocellulose. Western blotting was carried out with an anti-103P2D6 rabbit pAb (2 μg/ml) raised against a peptide encoding amino acids 163-176 in the 103P2D6 extracellular domain. Anti-103P2D6 immunoreactive bands were detected by incubation with anti-rabbit-HRP conjugated secondary Ab and developed using enhanced chemiluminescence and exposure to autoradiographic film. Indicated by arrow is a specific anti-103P2D6 immunoreactive band of approximately 85 kD in 103P2D6-transfected cells but not in control cells. For the data in panel B, 103P2D6 transfected and vector transfected cells were stained with 10 μg/ml of anti-103P2D6 pAb and subjected to flow cytometry following incubation with anti-rabbit-FITC conjugated secondary Ab. Shown is a fluorescent shift in 103P2D6-transfected cells compared to the vector transfected cells, indicating cell surface expression of 103P2D6 protein.

[0025]FIG. 13 shows the expression of 103P2D6 as assayed in a panel of human cancers (T) and their respective matched normal tissues (N) on RNA dot blots. 103P2D6 expression was seen in cancers of the kidney, breast, prostate, uterus, ovary, cervix, colon, stomach and rectum. 103P2D6 was also found to be highly expressed in the two human cancer cell lines, the CML line K562 and the colorectal carcinoma SW480. The expression detected in normal adjacent tissues (isolated from diseased tissues) but not in normal tissues, isolated from healthy donors, indicates that these tissues are not fully normal and that 103P2D6 may be expressed in early stage tumors and that it has utility as a diagnostic marker. Cancer cell lines are, from left to right, HeLa (cervical carcinoma); Daudi (Burkiff's lymphoma); K562 (CML); HL-60 (PML); G361 (melanoma); A549 (lung carcinoma); MOLT-4 (lymphoblastic leuk.); SW480 (colorectal carcinoma); Raji (Burkitt's lymphoma).

[0026]FIG. 14 shows data where RNA was isolated from prostate tumors (T) and their adjacent normal tissues (N) obtained from the following prostate cancer patients (Pt); patient 1, Gleason score 4+5; patient 2, Gleason score 3+4; and, patient 3, Gleason score 4+3. NP=normal prostate. Northern analysis was performed using 10 μg of total RNA for each sample. Expression of 103P2D6 was seen in all three tumor samples tested and their respective normal tissues.

[0027]FIG. 15 provides data for Northern experiments where RNA was isolated from kidney tumors (T) and their adjacent normal tissues (N) obtained from the following kidney cancer patients: Patient 1-Papillary Type, Stage I, Grade 214; Patient 2—Invasive papillary carcinoma, Grade 2/4; Patient 3—Clear cell type Grade 1/3, focally 2/3; Patient 4—Clear cell type, stage III, Grade 2/4; Patient 5—Clear cell type, stage III, Grade 3/4; Patient 6—Clear cell type, stage III, Grade 3/4; Patient 7—Clear cell type, Grade III. CL=Cell lines (from left to right): 769-P, A498, SW839; NK=Normal kidney; N=Normal adjacent tissue; T=Tumor. The Northern analysis was performed using long of total RNA for each sample. Elevated expression of 103P2D6 was observed in kidney tumors and normal adjacent tissues isolated from kidney cancer patients as compared to normal kidney.

[0028]FIG. 16 shows the results of Northern analysis where RNA was isolated from bladder cancers and adjacent normal tissue obtained from bladder cancer patients. The Northern analysis was performed using 10 μg of total RNA for each sample. Expression of 103P2D6 was seen in bladder tumor but not in normal adjacent tissue. N_(at)=Normal adjacent tissue; T=Tumor.

DETAILED DESCRIPTION OF THE INVENTION

[0029] I.) Definitions:

[0030] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

[0031] As used herein, the terms “advanced prostate cancer”, “locally advanced prostate cancer”, “advanced disease” and “locally advanced disease” mean prostate cancers that have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Association (AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients having clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the prostate base. Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles.

[0032] “Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence 103P2D6 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence 103P2D6. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.

[0033] The term “analog” refers to a molecule that is structurally similar or shares similar or corresponding attributes with another molecule (e.g. a 103P2D6-related protein). For example an analog of the 103P2D6 protein can be specifically bound by an antibody or T cell that specifically binds to 103P2D6.

[0034] The term “antibody” is used in the broadest sense. Therefore an “antibody” can be naturally occurring or man-made such as monoclonal antibodies produced by conventional hybridoma technology. Anti-103P2D6 antibodies comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domain and/or one or more complementarity determining regions of these antibodies.

[0035] As used herein, an “antibody fragment” is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen-binding region. In one embodiment it specifically covers single anti-103P2D6 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-103P2D6 antibody compositions with polyepitopic specificity.

[0036] The term “codon optimized sequences” refers to nucleotide sequences that have been optimized for a particular host species by replacing any codons having a usage frequency of less than about 20%. Nucleotide sequences that have been optimized for expression in a given host species by elimination of spurious polyadenylation sequences, elimination of exon/intron splicing signals, elimination of transposon-like repeats and/or optimization of GC content in addition to codon optimization are referred to herein as an “expression enhanced sequences.”

[0037] The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to maytansinoids, ytrium, bismuth ricin, ricin A-chain, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes of Lu. Antibodies may also be conjugated to an anti-cancer pro-drug activating enzyme capable of converting the pro-drug to its active form.

[0038] The term “homolog” refers to a molecule which exhibits homology to another molecule, by for example, having sequences of chemical residues that are the same or similar at corresponding positions.

[0039] As used herein, the terms “hybridize”, “hybridizing”, “hybridizes” and the like, used in the context of polynucleotides, are meant to refer to conventional hybridization conditions, preferably such as hybridization in 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures for hybridization are above 37 degrees C. and temperatures for washing in 0.1×SSC/0.1% SDS are above 55 degrees C.

[0040] As used herein, a polynucleotide is said to be “isolated” when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than the 103P2D6 gene or that encode polypeptides other than 103P2D6 gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 103P2D6 polynucleotide.

[0041] As used herein, a protein is said to be “isolated” when physical, mechanical or chemical methods are employed to remove the 103P2D6 protein from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 103P2D6 protein. Alternatively, an isolated protein can be prepared by chemical means.

[0042] The term “manmal” as used herein refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.

[0043] As used herein, the terms “metastatic prostate cancer” and “metastatic disease” mean prostate cancers that have spread to regional lymph nodes or to distant sites, and are meant to include stage D disease under the AUA system and stage T×N×M+under the TNM system. As is the case with locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred treatment modality. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment initiation. Approximately half of these androgen-refractory patients die within 6 months after developing that status. The most common site for prostate cancer metastasis is bone. Prostate cancer bone metastases are often osteoblastic rather than osteolytic (i.e., resulting in net bone formation). Bone metastases are found most frequently in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, liver and brain. Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.

[0044] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts.

[0045] As used herein “motif” as in biological motif of an 103P2D6-related protein, refers to any set of amino acids forming part of the primary sequence of a protein, either contiguous or capable of being aligned to certain positions that are generally invariant, that is associated with a particular function (e.g. protein-protein interaction, protein-DNA interaction, etc) or modification (e.g. that is phosphorylated, glycosylated or aridated), or localization (e.g. secretory sequence, nuclear localization sequence, etc.) or a sequence that is correlated with being immunogenic, either humorally or cellularly.

[0046] As used herein, the term “polynucleotide” means a polymeric form of nucleotides of at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA and/or RNA. In the art, this term if often used interchangeably with “oligonucleotide”. A polynucleotide can comprise a nucleotide sequence disclosed herein wherein thymidine (T) (as shown for example in SEQ ID NO: 1) can also be uracil (U); this definition pertains to the differences between the chemical structures of DNA and RNA, in particular the observation that one of the four major bases in RNA is uracil (U) instead of thymidine (T).

[0047] As used herein, the term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, standard three letter or single letter designations for amino acids are used. In the art, this term is often used interchangeably with “peptide” or “protein”.

[0048] As used herein, a “recombinant” DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro.

[0049] “Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

[0050] “Stringent conditions” or “high stringency conditions”, as defined herein, are identified by, but not limited to, those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 nM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (PH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium. citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C. “Moderately stringent conditions” are described by, but not limited to, those in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

[0051] A “transgenic animal” (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A “transgene” is a DNA that is integrated into the genome of a cell from which a transgenic animal develops.

[0052] The term “variant” refers to a molecule that exhibits a variation from a described type or norm, such as a protein that has one or more different amino acid residues in the corresponding position(s) of a specifically described protein (e.g. the 103P2D6 protein shown in FIG. 2 and FIG. 3). An analog is an example of a variant protein.

[0053] As used herein, the 103P2D6-related gene and 103P2D6-related protein includes the 103P2D6 genes and proteins specifically described herein, as well as structurally and/or functionally similar variants or analog of the foregoing. 103P2D6 peptide analogs generally share at least about 50%, 60%, 70%, 80%, 90% or more amino acid homology (using BLAST criteria). 103P2D6 nucleotide analogs preferably share 50%, 60%, 70%, 80%, 90% or more nucleic acid homology (using BLAST criteria). In some embodiments, however, lower homology is preferred so as to select preferred residues in view of species-specific codon preferences for optimized protein expression and production and/or immunogenicity-modulated peptide epitopes tailored to a particular target population, e.g. HLA type, as is appreciated by those skilled in the art.

[0054] The 103P2D6-related proteins of the invention include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art. Fusion proteins that combine parts of different 103P2D6 proteins or fragments thereof, as well as fusion proteins of a 103P2D6 protein and a heterologous polypeptide are also included. Such 103P2D6 proteins are collectively referred to as the 103P2D6-related proteins, the proteins of the invention, or 103P2D6. As used herein, the term “103P2D6-related protein” refers to a polypeptide fragment or an 103P2D6 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids.

[0055] II.) Properties of 103P2D6.

[0056] As disclosed herein, 103P2D6 exhibits specific properties that are analogous to those found in a family of molecules whose polynucleotides, polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) and anti-polypeptide antibodies are used in well known diagnostic assays that examine conditions associated with dysregulated cell growth such as cancer, in particular prostate cancer (see, e.g., both its highly specific pattern of tissue expression as well as its overexpression in prostate cancers as described for example in Example 3). The best-known member of this class is PSA, the archetypal marker that has been used by medical practitioners for years to identify and monitor the presence of prostate cancer (see, e.g., Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J. Urol. August;162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19): 1635-1640(1999)). A variety of other diagnostic markers are also used in this context including p53 and K-ras (see, e.g., Tulchinsky et al., Int J Mol Med July 1999;4(1):99-102 and Minimoto et al., Cancer Detect Prev 2000;24(1):1-12). Therefore, this disclosure of the 103P2D6 polynucleotides and polypeptides (as well as the 103P2D6 polynucleotide probes and anti-103P2D6 antibodies used to identify the presence of these molecules) and their properties allows skilled artisans to utilize these molecules in methods that are analogous to those used, for example, in a variety of diagnostic assays directed to examining conditions associated with cancer.

[0057] Typical embodiments of diagnostic methods that utilize the 103P2D6 polynucleotides, polypeptides, reactive T cells and antibodies are analogous to those methods from well-established diagnostic assays that employ, e.g., PSA polynucleotides, polypeptides, reactive T cells and antibodies. For example, just as PSA polynucleotides are used as probes (for example in Northern analysis, see, e.g., Sharief et al., Biochem. Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example in PCR analysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190 (2000)) to observe the presence and/or the level of PSA mRNAs in methods of monitoring PSA overexpression or the metastasis of prostate cancers, the 103P2D6 polynucleotides described herein can be utilized in the same way to detect 103P2D6 overexpression or the metastasis of prostate and other cancers expressing this gene. Alternatively, just as PSA polypeptides are used to generate antibodies specific for PSA which can then be used to observe the presence and/or the level of PSA proteins in methods to monitor PSA protein overexpression (see, e.g., Stephan et al., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the 103P2D6 polypeptides described herein can be utilized to generate antibodies for use in detecting 103P2D6 overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene.

[0058] Specifically, because metastases involves the movement of cancer cells from an organ of origin (such as the lung or prostate gland etc.) to a different area of the body (such as a lymph node), assays which examine a biological sample for the presence of cells expressing 103P2D6 polynucleotides and/or polypeptides can be used to provide evidence of metastasis. For example, when a biological sample from tissue that does not normally contain 103P2D6-expressing cells (lymph node) is found to contain 103P2D6-expressing cells such as the 103P2D6 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.

[0059] Alternatively 103P2D6 polynucleotides and/or polypeptides can be used to provide evidence of cancer, for example, when cells in a biological sample that do not normally express 103P2D6 or express 103P2D6 at a different level are found to express 103P2D6 or have an increased expression of 103P2D6 (see, e.g., the 103P2D6 expression in kidney, lung and colon cancer cells and in patient samples etc. shown in FIGS. 4-10). In such assays, artisans may further wish to generate supplementary evidence of metastasis by testing the biological sample for the presence of a second tissue restricted marker (in addition to 103P2D6) such as PSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)).

[0060] Just as PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, 103P2D6 polynucleotide fragments and polynucleotide variants are used in an analogous manner. In particular, typical PSA polynucleotides used in methods of monitoring PSA are probes or primers that consist of fragments of the PSA cDNA sequence. Illustrating this, primers used to PCR amplify a PSA polynucleotide must include less than the whole PSA sequence to function in the polymerase chain reaction. In the context of such PCR reactions, skilled artisans generally create a variety of different polynucleotide fragments that can be used as primers in order to amplify different portions of a polynucleotide of interest or to optimize amplification reactions (see, e.g., Caetano-Anolles, G. Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., Methods Mol. Biol. 98:121-154 (1998)). An additional illustration of the use of such fragments is provided in Example 3, where a 103P2D6 polynucleotide fragment is used as a probe to show the expression of 103P2D6 RNAs in cancer cells. In addition, variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, e.g., Sawai et al., Fetal Diagn. Ther. November-December 1996;11(6):407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubul et al. eds., 1995)). Polynucleotide fragments and variants are useful in this context where they are capable of binding to a target polynucleotide sequence (e.g. the 103P2D6 polynucleotide shown in SEQ ID NO: 1) under conditions of high stringency.

[0061] Furthermore, PSA polypeptides which contain an epitope that can be recognized by an antibody or T cell that specifically binds to that epitope are used in methods of monitoring PSA. 103P2D6 polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner. This practice of using polypeptide fragments or polypeptide variants to generate antibodies (such as anti-PSA antibodies or T cells) is typical in the art with a wide variety of systems such as fusion proteins being used by practitioners (see, e.g., Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995). In this context, each epitope(s) functions to provide the architecture with which an antibody or T cell is reactive. Typically, skilled artisans create a variety of different polypeptide fragments that can be used in order to generate immune responses specific for different portions of a polypeptide of interest (see, e.g., U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it may be preferable to utilize a polypeptide comprising one of the 103P2D6 biological motifs discussed herein or available in the art. Polypeptide fragments, variants or analogs are typically useful in this context as long as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence (e.g. the 103P2D6 polypeptide shown in SEQ ID NO: 2).

[0062] As shown herein, the 103P2D6 polynucleotides and polypeptides (as well as the 103P2D6 polynucleotide probes and anti-103P2D6 antibodies or T cells used to identify the presence of these molecules) exhibit specific properties that make them useful in diagnosing cancers of the prostate. Diagnostic assays that measure the presence of 103P2D6 gene products, in order to evaluate the presence or onset of a disease condition described herein, such as prostate cancer, are used to identify patients for preventive measures or further monitoring, as has been done so successfully with PSA. Moreover, these materials satisfy a need in the art for molecules having similar or complementary characteristics to PSA in situations where, for example, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as 103P2D6 polynucleotides and polypeptides (as well as the 103P2D6 polynucleotide probes and anti-103P2D6 antibodies used to identify the presence of these molecules) must be employed to confirm metastases of prostatic origin.

[0063] Finally, in addition to their use in diagnostic assays, the 103P2D6 polynucleotides disclosed herein have a number of other specific utilities such as their use in the identification of oncogenetic associated chromosomal abnormalities in 2q34, the chromosomal region to which the 103P2D6 gene maps (see Example 7 below). Moreover, in addition to their use in diagnostic assays, the 103P2D6-related proteins and polynucleotides disclosed herein have other utilities such as their use in the forensic analysis of tissues of unknown origin (see, e.g., Takahama K Forensic Sci Int Jun. 28, 1996;80(1-2): 63-9).

[0064] Additionally, 103P2D6-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 103P2D6. For example, the amino acid or nucleic acid sequence of FIG. 2, or fragments thereof, can be used to generate an immune response to the 103P2D6 antigen. Antibodies or other molecules that react with 103P2D6 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.

[0065] III.) 103P2D6 Polynucleotides

[0066] One aspect of the invention provides polynucleotides corresponding or complementary to all or part of an 103P2D6 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding an 103P2D6-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to an 103P2D6 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to an 103P2D6 gene, mRNA, or to an 103P2D6 encoding polynucleotide (collectively, “103P2D6 polynucleotides”). In all instances when referred to in this section, T can also be U in FIG. 2.

[0067] Embodiments of a 103P2D6 polynucleotide include: a 103P2D6 polynucleotide having the sequence shown in FIG. 2, the nucleotide sequence of 103P2D6 as shown in FIG. 2, wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2 where T is U. Further 103P2D6 nucleotides comprise, where T can be U:

[0068] (a) at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2, from nucleotide residue number 1 through nucleotide residue number 804; or,

[0069] (b) at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2, from nucleotide residue number 977 through nucleotide residue number 1036; or,

[0070] (c) at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2, from nucleotide residue number 1414 through nucleotide residue number 1815; or

[0071] (d) a polynucleotide whose starting base is in the range of 1-804 of FIG. 2 and whose ending base is in the range of 805-2493 of FIG. 2; or

[0072] (e) a polynucleotide whose starting base is in the range of 977-1036 of FIG. 2 and whose ending base is in the range of 1037-2493 of FIG. 2; or

[0073] (f) a polynucleotide whose starting base is in the range of 1414-1815 of FIG. 2 and whose ending base is in the range of 1816-2493 of FIG. 2; or

[0074] (g) a polynucleotide whose starting base is in the range of 805-976 of FIG. 2 and whose ending base is in the range of 977-2493 of FIG. 2; or

[0075] (h) a polynucleotide whose starting base is in the range of 805-2493 of FIG. 2 and whose ending base is in the range of 2494-4727 of FIG. 2; or

[0076] (i) a polynucleotide of (d-g) that is at least 10 nucleotide bases in length; or

[0077] (j) a polynucleotide that selectively hybridizes under stringent conditions to a polynucleotide of (a)-(g);

[0078] wherein a range is understood to specifically disclose all whole unit positions thereof. Moreover, a peptide that is encoded by any of the foregoing is also within the scope of the invention.

[0079] Also within the scope of the invention is a nucleotide, as well as any peptide encoded thereby, that starts at any of the following positions or ranges, and ends at a higher position or range: 1, 804, a range of 1-804, 805, a range of 805-976; a range of 805-2493; a range of 977-1036, a range of 1037-1413; a range of 1414-1815; a range of 1816-2493; a range of 2494-4727; wherein a range as used in this section is understood to specifically disclose all whole unit positions thereof.

[0080] Another embodiment of the invention comprises a polynucleotide that encodes a 103P2D6-related protein whose sequence is encoded by the cDNA contained in the plasmids deposited with American Type Culture Collection as Accession No. PTA-1155 or PTA-1895. Another embodiment comprises a polynucleotide that hybridizes under stringent hybridization conditions, to the human 103P2D6 cDNA shown in SEQ ID NO: 1 or to a polynucleotide fragment thereof.

[0081] Typical embodiments of the invention disclosed herein include 103P2D6 polynucleotides that encode specific portions of the 103P2D6 mRNA sequence (and those which are complementary to such sequences) such as those that encode the protein and fragments thereof, for example of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids.

[0082] For example, representative embodiments of the invention disclosed herein include: polynucleotides and their encoded peptides themselves encoding about amino acid 1 to about amino acid 10 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the 103P2D6 protein shown in FIG. 2 and FIG. 3 and polynucleotides encoding about amino acid 90 to about amino acid 100 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, in increments of about 10 amino acids, ending at amino acid 532. Accordingly polynucleotides encoding portions of the amino acid sequence (of about 10 amino acids), of amino acids 100-532 of the 103P2D6 protein are embodiments of the invention. Wherein it is understood that each particular amino acid position discloses that position plus or minus five amino acid residues.

[0083] Polynucleotides encoding relatively long portions of the 103P2D6 protein are also within the scope of the invention. Additional illustrative embodiments of the invention disclosed herein include 103P2D6 polynucleotide fragments encoding one or more of the biological motifs contained within the 103P2D6 protein sequence, including one or more of the motif-bearing subsequences of the 103P2D6 protein set forth in Table XIX. In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of 103P2D6 that exhibit homology to a known molecule. In another embodiment of the invention, typical polynucleotide fragments can encode one or more of the 103P2D6 N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.

[0084] III.A.) Uses of 103P2D6 Polynucleotides

[0085] III.A.1.) Monitoring of Genetic Abnormalities

[0086] The polynucleotides of the preceding paragraphs have a number of different specific uses. The human 103P2D6 gene maps to chromosome 4p12-p14 as determined using the GeneBridge4 radiation hybrid panel (see Example 7). For example, because the 103P2D6 gene maps to chromosome 4p12-p14, polynucleotides that encode different regions of the 103P2D6 protein are used to characterize cytogenetic abnormalities on chromosome 4, band p12-p14 that have been identified as being associated with various cancers. In particular, a variety of chromosomal abnormalities in 4p12-p14 including translocations and deletions have been identified as frequent cytogenetic abnormalities in a number of different cancers (see, e.g., Zimonjic, D. B. et al., 1999, Hepatology 29(4):1208-14; Wu, X. et al., 1995, Cancer Res. 55(3):557-61; Arribas, R. et al., 1999, Lab. Invest. 79(2):111-22). Thus, polynucleotides encoding specific regions of the 103P2D6 protein provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in this region of chromosome 2 that may contribute to the malignant phenotype. In this context, these polynucleotides satisfy a need in the art for expanding the sensitivity of chromosomal screening in order to identify more subtle and less common chromosomal abnormalities (see e.g. Evans et al., Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).

[0087] Furthermore, as 103P2D6 was shown to be highly expressed in prostate and other cancers, 103P2D6 polynucleotides are used in methods assessing the status of 103P2D6 gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the 103P2D6 protein are used to assess the presence of perturbations (such as deletions, insertions, point mutations, or alterations resulting in a loss of an antigen etc.) in specific regions of the 103P2D6 gene, such as such regions containing one or more motifs. Exemplary assays include both RT-PCR assays as well as single-strand conformation polymorphism (SSCP) analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8): 369-378 (1999), both of which utilize polynucleotides encoding specific regions of a protein to examine these regions within the protein.

[0088] III.A.2.) Antisense Embodiments

[0089] Other specifically contemplated nucleic acid related embodiments of the invention disclosed herein are genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nucleic acid molecules based on an alternative backbone, or including alternative bases, whether derived from natural sources or synthesized, and include molecules capable of inhibiting the RNA or protein expression of 103P2D6. For example, antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives, that specifically bind DNA or RNA in a base pair-dependent manner. A skilled artisan can readily obtain these classes of nucleic acid molecules using the 103P2D6 polynucleotides and polynucleotide sequences disclosed herein.

[0090] Antisense technology entails the administration of exogenous oligonucleotides that bind to a target polynucleotide located within the cells. The term “antisense” refers to the fact that such oligonucleotides are complementary to their intracellular targets, e.g., 103P2D6. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 103P2D6 antisense oligonucleotides of the present invention include derivatives such as S-oligonucleotides (phosphorothioate derivatives or 8-oligos, see, Jack Cohen, supra), which exhibit enhanced cancer cell growth inhibitory action. S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of an oligonucleotide (O-oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos of the present invention can be prepared by treatment of the corresponding O-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer reagent. See Iyer, R. P. et al, J. Org. Chem. 55:4693-4698 (1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990). Additional 103P2D6 antisense oligonucleotides of the present invention include morpholino antisense oligonucleotides known in the art (see, e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6: 169-175).

[0091] The 103P2D6 antisense oligonucleotides of the present invention typically can be RNA or DNA that is complementary to and stably hybridizes with the first 100 5′ codons or last 100 3′ codons of the 103P2D6 genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although high degrees of complementarity are preferred. Use of an oligonucleotide complementary to this region allows for the selective hybridization to 103P2D6 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 103P2D6 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 103P2D6 mRNA. Optionally, 103P2D6 antisense oligonucleotide is a 30-mer oligonucleotide that is complementary to a region in the first 10 5′ codons or last 10 3′ codons of 103P2D6. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of 103P2D6 expression, see, e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

[0092] III.A.3.) Primers and Primer Pairs

[0093] Further specific embodiments of this nucleotides of the invention include primers and primer pairs, which allow the specific amplification of polynucleotides of the invention or of any specific parts thereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the invention or to any part thereof Probes can be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme. Such probes and primers are used to detect the presence of a 103P2D6 polynucleotide in a sample and as a means for detecting a cell expressing a 103P2D6 protein.

[0094] Examples of such probes include polypeptides comprising all or part of the human 103P2D6 cDNA sequences shown in FIG. 2. Examples of primer pairs capable of specifically amplifying 103P2D6 mRNAs are also described in the Examples. As will be understood by the skilled artisan, a great many different primers and probes can be prepared based on the sequences provided herein and used effectively to amplify and/or detect a 103P2D6 mRNA.

[0095] The 103P2D6 polynucleotides of the invention are useful for a variety of purposes, including but not limited to their use as probes and primers for the amplification and/or detection of the 103P2D6 gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of 103P2D6 polypeptides; as tools for modulating or inhibiting the expression of the 103P2D6 gene(s) and/or translation of the 103P2D6 transcript(s); and as therapeutic agents.

[0096] III.A.4.) Isolation of 103P2D6-Encoding Nucleic Acid Molecules

[0097] The 103P2D6 cDNA sequences described herein enable the isolation of other polynucleotides encoding 103P2D6 gene product(s), as well as the isolation of polynucleotides encoding 103P2D6 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of the 103P2D6 gene product as well as polynucleotides that encode analogs of 103P2D6-related proteins. Various molecular cloning methods that can be employed to isolate fall length cDNAs encoding an 103P2D6 gene are well known (See, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2d edition., Cold Spring Harbor Press, New York, 1989; Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley and Sons, 1995). For example, lambda phage cloning methodologies can be conveniently employed, using commercially available cloning systems (e.g., Lambda ZAP Express, Stratagene). Phage clones containing 103P2D6 gene cDNAs can be identified by probing with a labeled 103P2D6 cDNA or a fragment thereof For example, in one embodiment, the 103P2D6 cDNA (FIG. 2) or a portion thereof can be synthesized and used as a probe to retrieve overlapping and full-length cDNAs corresponding to a 103P2D6 gene. The 103P2D6 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 103P2D6 DNA probes or primers.

[0098] III.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

[0099] The invention also provides recombinant DNA or RNA molecules containing an 103P2D6 polynucleotide, fragment, analog or homologue thereof, including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known in the art, and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for generating such molecules are well known (see, for example, Sambrook et al, 1989, supra).

[0100] The invention further provides a host-vector system comprising a recombinant DNA molecule containing a 103P2D6 polynucleotide, fragment, analog or homologue thereof within a suitable prokaryotic or eukaryotic host cell. Examples of suitable eukaryotic host cells include a yeast cell, a plant cell, or an animal cell, such as a mammalian cell or an insect cell (e.g., a baculovirus-infectible cell such as an Sf9 or HighFive cell). Examples of suitable mammalian cells include various prostate cancer cell lines such as DU145 and TsuPr1, other transfectable or transducible prostate cancer cell lines, primary cells (PrEC), as well as a number of mammalian cells routinely used for the expression of recombinant proteins (e.g., COS, CHO, 293, 293T cells). More particularly, a polynucleotide comprising the coding sequence of 103P2D6 or a fragment, analog or homolog thereof can be used to generate 103P2D6 proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.

[0101] A wide range of host-vector systems suitable for the expression of 103P2D6 proteins or fragments thereof are available, see for example, Sambrook et al., 1989, supra; Current Protocols in Molecular Biology, 1995, supra). Preferred vectors for mammalian expression include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using these expression vectors, 103P2D6 can be expressed in several prostate cancer and non-prostate cell lines, including for example 293, 293T, rat-1, NIH 3T3 and TsuPr1. The host-vector systems of the invention are useful for the production of a 103P2D6 protein or fragment thereof Such host-vector systems can be employed to study the functional properties of 103P2D6 and 103P2D6 mutations or analogs.

[0102] Recombinant human 103P2D6 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 103P2D6-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 103P2D6 or fragment, analog or homolog thereof, the 103P2D6 or related protein is expressed in the 293T cells, and the recombinant 103P2D6 protein is isolated using standard purification methods (e.g., affinity purification using anti-103P2D6 antibodies). In another embodiment, a 103P2D6 coding sequence is subcloned into the retroviral vector pSRαMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 and rat-1 in order to establish 103P2D6 expressing cell lines. Various other expression systems well known in the art can also be employed. Expression constructs encoding a leader peptide joined in frame to the 103P2D6 coding sequence can be used for the generation of a secreted form of recombinant 103P2D6 protein.

[0103] As discussed herein, redundancy in the genetic code permits variation in 103P2D6 gene sequences. In particular, it is known in the art that specific host species often have specific codon preferences, and thus one can adapt the disclosed sequence as preferred for a desired host. For example, preferred analog codon sequences typically have rare codons (i.e., codons having a usage frequency of less than about 20% in known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific species are calculated, for example, by utilizing codon usage tables available on the INTERNET such as: http://www.dna.affrc.go.jp/˜nakamura/codon.html.

[0104] Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and/or other such well-characterized sequences that are deleterious to gene expression. The GC content of the sequence is adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. Other useful modifications include the addition of a translational initiation consensus sequence at the start of the open reading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080 (1989). Skilled artisans understand that the general rule that eukaryotic ribosomes initiate translation exclusively at the 5′ proximal AUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS 92(7): 2662-2666, (1995) and KozakNAR 15(20): 8125-8148 (1987)).

[0105] IV.) 103P2D6-Related Proteins

[0106] Another aspect of the present invention provides 103P2D6-related proteins. Specific embodiments of 103P2D6 proteins comprise a polypeptide having all or part of the amino acid sequence of human 103P2D6 as shown in FIG. 2. Alternatively, embodiments of 103P2D6 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 103P2D6 shown in FIG. 2.

[0107] In general, naturally occurring allelic variants of human 103P2D6 share a high degree of structural identity and homology (e.g., 90% or more homology). Typically, allelic variants of the 103P2D6 protein contain conservative amino acid substitutions within the 103P2D6 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 103P2D6. One class of 103P2D6 allelic variants are proteins that share a high degree of homology with at least a small region of a particular 103P2D6 amino acid sequence, but further contain a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift. In comparisons of protein sequences, the terms, similarity, identity, and homology each have a distinct meaning as appreciated in the field of genetics. Moreover, orthology and paralogy can be important concepts describing the relationship of members of a given protein family in one organism to the members of the same family in other organisms.

[0108] Amino acid abbreviations are provided in Table II. Conservative amino acid substitutions can frequently be made in a protein without altering either the conformation or the function of the protein. Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered “conservative” in particular environments (see, e.g. Table III herein; pages 13-15 “Biochemistry” 2^(nd) ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS 1992 Vol. 89 10915-10919; Lei et al., J Biol Chem May 19, 1995; 270(20):11882-6).

[0109] Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 103P2D6 proteins such as polypeptides having amino acid insertions, deletions and substitutions. 103P2D6 variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13.4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the cloned DNA to produce the 103P2D6 variant DNA.

[0110] Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence that is involved in a specific biological activity such as a protein-protein interaction. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions (Creighton, The Proteins, (W. H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yield adequate amounts of variant, an isosteric amino acid can be used.

[0111] As defined herein, 103P2D6 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is “cross reactive” with a 103P2D6 protein having the amino acid sequence of SEQ ID NO: 2. As used in this sentence, “cross reactive” means that an antibody or T cell that specifically binds to an 103P2D6 variant also specifically binds to the 103P2D6 protein having the amino acid sequence of SEQ ID NO: 2. A polypeptide ceases to be a variant of the protein shown in SEQ ID NO: 2 when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the 103P2D6 protein. Those skilled in the art understand that antibodies that recognize proteins bind to epitopes of varying size, and a grouping of the order of about four or five amino acids, contiguous or not, is regarded as a typical number of amino acids in a minimal epitope. See, e.g., Nair et al., J. Immunol 2000 165(12): 6949-6955; Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985) 135(4):2598-608.

[0112] Another class of 103P2D6-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with the amino acid sequence of SEQ ID NO: 2 or a fragment thereof. Another specific class of 103P2D6 protein variants or analogs comprise one or more of the 103P2D6 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 103P2D6 fragments (nucleic or amino acid) that have altered functional (e.g. immunogenic) properties relative to the starting fragment. It is to be appreciated that motifs now or which become part of the art are to be applied to the nucleic or amino acid sequences of FIG. 2.

[0113] As discussed herein, embodiments of the claimed invention include polypeptides containing less than the 532 amino acid sequence of the 103P2D6 protein shown in FIG. 2. For example, representative embodiments of the invention comprise peptides/proteins having any 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of the 103P2D6 protein shown in FIG. 2 and FIG. 3.

[0114] Moreover, representative embodiments of the invention disclosed herein include polypeptides consisting of about amino acid 1 to about amino acid 10 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of the 103P2D6 protein shown in FIG. 2 and FIG. 3 and polypeptides consisting of about amino acid 90 to about amino acid 100 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, etc. throughout the entirety of the 103P2D6 sequence. Further, this definition defines polypeptides consisting of 10 amino acid stretches of the amino acid sequence of amino acids 100-532 of the 103P2D6 protein. Moreover, polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.) of the 103P2D6 protein shown in FIG. 2 and FIG. 3 are embodiments of the invention. It is to be appreciated that the starting and stopping positions in this paragraph refer to the specified position as well as that position plus or minus 5 residues.

[0115] 103P2D6-related proteins are generated using standard peptide synthesis technology or using chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a 103P2D6related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of the 103P2D6 protein (or variants, homologs or analogs thereof).

[0116] IV.A.) Motif-Bearing Protein Embodiments

[0117] Additional illustrative embodiments of the invention disclosed herein include 103P2D6 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within the 103P2D6 polypeptide sequence set forth in FIG. 2 or FIG. 3. Various motifs are known in the art, and a protein can be evaluated for the presence of such motifs by a number of publicly available sites (see, e.g.: http://pfam.wustl.edu/; http://searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html http://psort.ims.u-tokyo.ac.jp/; http://www.cbs.dtu.dk/; http://www.ebi.ac.uk/interpro/scan.html; http://www.expasy.ch/tools/scripsit1.html; Epimatrix™ and Epimer™, Brown University, http://www.brown.edu/Research/TB-HIV_Lab/epiatrix/epimatrix.html; and BIMAS, http://bimas.dert.nih.gov/.).

[0118] Motif bearing subsequences of the 103P2D6 protein are set forth and identified in Table XIX.

[0119] Table XX sets forth several frequently occurring motifs based on pfam searches (http://pfam.wustl.edu/). The columns of Table XX list (1) motif name abbreviation, (2) percent identity found amongst the different member of the motif family, (3) motif name or description and (4) most common function; location information is included if the motif is relevant for location.

[0120] Polypeptides comprising one or more of the 103P2D6 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 103P2D6 motifs discussed above are associated with growth dysregulation and because 103P2D6 is overexpressed in certain cancers (See, e.g., Table I). Casein kinase II, cAMP and cCMP-dependent protein kinase, and Protein Kinase C, for example, are enzymes known to be associated with the development of the malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2): 165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both glycosylation and myristoylation are protein modifications also associated with cancer and cancer progression (see e.g. Dennis et al., Biochem. Biophys. Acta 1473(1):21-34 (1999); Raju et al., Exp. Cell Res. 235(1): 145-154 (1997)). Amidation is another protein modification also associated with cancer and cancer progression (see e.g. Treston et al., J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)).

[0121] In another embodiment, proteins of the invention comprise one or more of the immunoreactive epitopes identified in accordance with art-accepted methods, such as the peptides set forth in Tables V-XVIII. CTL epitopes can be determined using specific algorithms to identify peptides within an 103P2D6 protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV (A) and Table IV (B); Epimatrix™ and Epimer™, Brown University, http://www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, http:/bimas.dcrt.nih.gov/. Moreover, processes for identifying peptides that have sufficient binding affinity for HLA molecules and which are correlated with being immunogenic epitopes, are well known in the art, and are carried out without undue experimentation. In addition, processes for identifying peptides that are immunogenic epitopes, are well known in the art, and are carried out without undue experimentation either in vitro or in vivo.

[0122] Also known in the art are principles for creating analogs of such epitopes in order to modulate immunogenicity. For example, one begins with an epitope that bears a CTL or HTL motif (see, e.g., the HLA Class I motifs or Table IV (A) and the HTL motif of Table IV (B)). The epitope is analoged by substituting out an amino acid at one of the specified positions, and replacing it with another amino acid specified for that position.

[0123] A variety of references reflect the art regarding the identification and generation of epitopes in a protein of interest as well as analogs thereof. See, for example, WO 9733602 to Chestnut et al.; Sette, Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001 166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondo et al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol. 1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994 152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3): 266-278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J. Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.

[0124] Related embodiments of the inventions include polypeptides comprising combinations of the different motifs set forth in Table XIX, and/or, one or more of the predicted CTL epitopes of Table V through Table XVIII, and/or, one or more of the T cell binding motifs known in the art. Preferred embodiments contain no insertions, deletions or substitutions either within the motifs or the intervening sequences of the polypeptides. In addition, embodiments which include a number of either N-terminal and/or C-terminal amino acid residues on either side of these motifs may be desirable (to, for example, include a greater portion of the polypeptide architecture in which the motif is located). Typically the number of N-terminal and/or C-terminal amino acid residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.

[0125] 103P2D6-related proteins are embodied in many forms, preferably in isolated form. A purified 103P2D6 protein molecule will be substantially free of other proteins or molecules that impair the binding of 103P2D6 to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 103P2D6-related proteins include purified 103P2D6-related proteins and functional, soluble 103P2D6-related proteins. In one embodiment, a functional, soluble 103P2D6 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.

[0126] The invention also provides 103P2D6 proteins comprising biologically active fragments of the 103P2D6 amino acid sequence shown in FIG. 2. Such proteins exhibit properties of the 103P2D6 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the 103P2D6 protein; to be bound by such antibodies; to elicit the activation of HTL or CTL; and/or, to be recognized by HTL or CTL.

[0127] 103P2D6-related polypeptides that contain particularly interesting structures can be predicted and/or identified using various analytical techniques well known in the art, including, for example, the methods of Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or on the basis of immunogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-103P2D6 antibodies, or T cells or in identifying cellular factors that bind to 103P2D6.

[0128] CTL epitopes can be determined using specific algorithms to identify peptides within an 103P2D6 protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV (A) and Table IV (B); Epimatrix™ and Epimer™, Brown University (http://www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and BIMAS, http://bimas.dcrt.nih.gov/). Illustrating this, peptide epitopes from 103P2D6 that are presented in the context of human MHC class I molecules HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (Tables V-XVIII). Specifically, the complete amino acid sequence of the 103P2D6 protein was entered into the HLA Peptide Motif Search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS) web site listed above. The HLA peptide motif search algorithm was developed by Dr. Ken Parker based on binding of specific peptide sequences in the groove of HLA Class I molecules and specifically HLA-A2 (see, e.g., Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994)). This algorithm allows location and ranking of 8-mer, 9-mer, and 10-mer peptides from a complete protein sequence for predicted binding to HLA-A2 as well as numerous other HLA Class I molecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers. For example, for class I HLA-A2, the epitopes preferably contain a leucine (L) or methionine (M) at position 2 and a valine (V) or leucine (L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7 (1992)). Selected results of 103P2D6 predicted binding peptides are shown in Tables V-XVIII herein. In Tables V-XVIII, the top 50 ranking candidates, 9-mers and 10-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. The binding score corresponds to the estimated half-time of dissociation of complexes containing the peptide at 37° C. at pH 6.5. Peptides with the highest binding score are predicted to be the most tightly bound to HLA Class I on the cell surface for the greatest period of time and thus represent the best immunogenic targets for T-cell recognition.

[0129] Actual binding of peptides to an HLA allele can be evaluated by stabilization of HLA expression on the antigen-processing defective cell line T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa et al., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides can be evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes (CTL) in the presence of antigen presenting cells such as dendritic cells.

[0130] It is to be appreciated that every epitope predicted by the BIMAS site, Epimer™ and Epimatrix™ sites, or specified by the HLA class I or class I motifs available in the art or which become part of the art such as set forth in Table IV (A) and Table IV (B) are to be “applied” to the 103P2D6 protein. As used in this context “applied” means that the 103P2D6 protein is evaluated, e.g., visually or by computer-based patterns finding methods, as appreciated by those of skill in the relevant art. Every subsequence of the 103P2D6 of 8, 9, 10, or 11 amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.

[0131] IV.B.) Expression of 103P2D6-Related Proteins

[0132] In an embodiment described in the examples that follow, 103P2D6 can be conveniently expressed in cells (such as 293T cells) transfected with a commercially available expression vector such as a CMV-driven expression vector encoding 103P2D6 with a C-terminal 6×His and MYC tag (pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, Nashville Tenn.). The Tag5 vector provides an IgGK secretion signal that can be used to facilitate the production of a secreted 103P2D6 protein in transfected cells. The secreted HIS-tagged 103P2D6 in the culture media can be purified, e.g., using a nickel column using standard techniques.

[0133] IV.C.) Modifications of 103P2D6-Related Proteins

[0134] Modifications of 103P2D6-related proteins such as covalent modifications are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a 103P2D6 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of the 103P2D6. Another type of covalent modification of the 103P2D6 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of a protein of the invention. Another type of covalent modification of 103P2D6 comprises linking the 103P2D6 polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0135] The 103P2D6-related proteins of the present invention can also be modified to form a chimeric molecule comprising 103P2D6 fused to another, heterologous polypeptide or amino acid sequence. Such a chimeric molecule can be synthesized chemically or recombinantly. A chimeric molecule can have a protein of the invention fused to another tumor-associated antigen or fragment thereof. Alternatively, a protein in accordance with the invention can comprise a fusion of fragments of the 103P2D6 sequence (amino or nucleic acid) such that a molecule is created that is not, through its length, directly homologous to the amino or nucleic acid sequences respectively of FIG. 2. Such a chimeric molecule can comprise multiples of the same subsequence of 103P2D6. A chimeric molecule can comprise a fusion of a 103P2D6-related protein with a polyhistidine epitope tag, which provides an epitope to which immobilized nickel can selectively bind. The epitope tag is generally placed at the amino- or carboxyl-terminus of the 103P2D6. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 103P2D6-related protein with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an “immnunoadhesin”), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a 103P2D6 polypeptide in place of at least one variable region within an Ig molecule. In a preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgGI molecule. For the production of immunoglobulin fusions see, e.g., U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

[0136] IV.D.) Uses of 103P2D6-Related Proteins

[0137] The proteins of the invention have a number of different specific uses. As 103P2D6 is highly expressed in prostate and other cancers, 103P2D6-related proteins are used in methods that assess the status of 103P2D6 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of the 103P2D6 protein are used to assess the presence of perturbations (such as deletions, insertions, point mutations etc.) in those regions (such as regions containing one or more motifs). Exemplary assays utilize antibodies or T cells targeting 103P2D6-related proteins comprising the amino acid residues of one or more of the biological motifs contained within the 103P2D6 polypeptide sequence in order to evaluate the characteristics of this region in normal versus cancerous tissues or to elicit an immune response to the epitope. Alternatively, 103P2D6-related proteins that contain the amino acid residues of one or more of the biological motifs in the 103P2D6 protein are used to screen for factors that interact with that region of 103P2D6.

[0138] 103P2D6 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of an 103P2D6 protein), for identifying agents or cellular factors that bind to 103P2D6 or a particular structural domain thereof, and in various therapeutic and diagnostic contexts, including but not limited to diagnostic assays, cancer vaccines and methods of preparing such vaccines.

[0139] Proteins encoded by the 103P2D6 genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents and cellular constituents that bind to an 103P2D6 gene product. Antibodies raised against an 103P2D6 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 103P2D6 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 103P2D6-related nucleic acids or proteins are also used in generating HTL or CTL responses.

[0140] Various immunological assays useful for the detection of 103P2D6 proteins are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods, and the like. Antibodies can be labeled and used as immunological imaging reagents capable of detecting 103P2D6-expressing cells (e.g., in radioscintigraphic imaging methods). 103P2D6 proteins are also particularly useful in generating cancer vaccines, as further described herein.

[0141] V.) 103P2D6 Antibodies

[0142] Another aspect of the invention provides antibodies that bind to 103P2D6-related proteins. Preferred antibodies specifically bind to a 103P2D6-related protein and do not bind (or bind weakly) to peptides or proteins that are not 103P2D6-related proteins. For example, antibodies bind 103P2D6 can bind 103P2D6-related proteins such as the homologs or analogs thereof.

[0143] 103P2D6 antibodies of the invention are particularly useful in prostate cancer diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodies are useful in the treatment, diagnosis, and/or prognosis of other cancers, to the extent 103P2D6 is also expressed or overexpressed in these other cancers. Moreover, intracellularly expressed antibodies (e.g., single chain antibodies) are therapeutically useful in treating cancers in which the expression of 103P2D6 is involved, such as advanced or metastatic prostate cancers.

[0144] The invention also provides various immunological assays useful for the detection and quantification of 103P2D6 and mutant 103P2D6-related proteins. Such assays can comprise one or more 103P2D6 antibodies capable of recognizing and binding a 103P2D6-related protein, as appropriate. These assays are performed within various immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

[0145] Immunological non-antibody assays of the invention also comprise T cell immunogenicity assays (inhibitory or stimulatory) as well as major histocompatibility complex (MHC) binding assays.

[0146] In addition, immunological imaging methods capable of detecting prostate cancer and other cancers expressing 103P2D6 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled 103P2D6 antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 103P2D6 expressing cancers such as prostate cancer.

[0147] 103P2D6 antibodies are also used in methods for purifying a 103P2D6-related protein and for isolating 103P2D6 homologues and related molecules. For example, a method of purifying a 103P2D6-related protein comprises incubating an 103P2D6 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 103P2D6-related protein under conditions that permit the 103P2D6 antibody to bind to the 103P2D6-related protein; washing the solid matrix to eliminate impurities; and eluting the 103P2D6-related protein from the coupled antibody. Other uses of the 103P2D6 antibodies of the invention include generating anti-idiotypic antibodies that mimic the 103P2D6 protein.

[0148] Various methods for the preparation of antibodies are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host using a 103P2D6-related protein, peptide, or fragment, in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, fusion proteins of 103P2D6 can also be used, such as a 103P2D6 GST-fusion protein. In a particular embodiment, a GST fusion protein comprising all or most of the amino acid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogen to generate appropriate antibodies. In another embodiment, a 103P2D6-related protein is synthesized and used as an immunogen.

[0149] In addition, naked DNA immunization techniques known in the art are used (with or without purified 103P2D6-related protein or 103P2D6 expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

[0150] The amino acid sequence of 103P2D6 as shown in FIG. 2 or FIG. 3 can be analyzed to select specific regions of the 103P2D6 protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of the 103P2D6 amino acid sequence are used to identify hydrophilic regions in the 103P2D6 structure. Regions of the 103P2D6 protein that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. Thus, each region identified by any of these programs or methods is within the scope of the present invention. Methods for the generation of 103P2D6 antibodies are further illustrated by way of the examples provided herein. Methods for preparing a protein or polypeptide for use as an immunogen are well known in the art. Also well known in the art are methods for preparing immunogenic conjugates of a protein with a carrier, such as BSA, KLH or other carrier protein. In some circumstances, direct conjugation using, for example, carbodumide reagents are used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, Ill., are effective. Administration of a 103P2D6 immunogen is often conducted by injection over a suitable time period and with use of a suitable adjuvant, as is understood in the art. During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.

[0151] 103P2D6 monoclonal antibodies can be produced by various means well known in the art. For example, immortalized cell lines that secrete a desired monoclonal antibody are prepared using the standard hybridoma technology of Kohler and Milstein or modifications that immortalize antibody-producing B cells, as is generally known. Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a 103P2D6-related protein. When the appropriate immortalized cell culture is identified, the cells can be expanded and antibodies produced either from in vitro cultures or from ascites fluid.

[0152] The antibodies or fragments of the invention can also be produced, by recombinant means. Regions that bind specifically to the desired regions of the 103P2D6 protein can also be produced in the context of chimeric or complementarity determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 103P2D6 antibodies can also be produced, and are preferred for use in therapeutic contexts. Methods for humanizing murine and other non-human antibodies, by substituting one or more of the non-human antibody CDRs for corresponding human antibody sequences, are well known (see for example, Jones et al., 1986, Nature 321: 522-525; Riechmnan et al., 1988, Nature 332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296.

[0153] Methods for producing fully human monoclonal antibodies include phage display and transgenic methods (for review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully human 103P2D6 monoclonal antibodies can be generated using cloning technologies employing large human Ig gene combinatorial libraries (i.e., phage display) (Griffiths and Hoogenboom, Building an in vitro immune system: human antibodies from phage display libraries. In: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries. Id., pp 65-82). Fully human 103P2D6 monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application WO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614; U.S. Pat. No. 6,162,963 issued Dec. 19, 2000; U.S. Pat. No. 6,150,584 issued Nov. 12, 2000; and, U.S. Pat. No. 6,114,598 issued Sep. 5, 2000). This method avoids the in vitro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.

[0154] Reactivity of 103P2D6 antibodies with an 103P2D6-related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 103P2D6-related proteins, 103P2D6-expressing cells or extracts thereof. A 103P2D6 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme. Further, bi-specific antibodies specific for two or more 103P2D6 epitopes are generated using methods generally known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

[0155] VI.) 103P2D6 Transgenic Animals

[0156] Nucleic acids that encode a 103P2D6-related protein can also be used to generate either transgenic animals or “knock out” animals which, in turn, are useful in the development and screening of therapeutically useful reagents. In accordance with established techniques, cDNA encoding 103P2D6 can be used to clone genomic DNA that encodes 103P2D6. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 103P2D6. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Pat. No. 4,736,866 issued Apr. 12, 1988, and U.S. Pat. No. 4,870,009 issued Sep. 26, 1989. Typically, particular cells would be targeted for 103P2D6 transgene incorporation with tissue-specific enhancers.

[0157] Transgenic animals that include a copy of a transgene encoding 103P2D6 can be used to examine the effect of increased expression of DNA that encodes 103P2D6. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this aspect of the invention, an animal is treated with a reagent and a reduced incidence of a pathological condition, compared to untreated animals that bear the transgene, would indicate a potential therapeutic intervention for the pathological condition.

[0158] Alternatively, non-human homologues of 103P2D6 can be used to construct a 103P2D6 “knock out” animal that has a defective or altered gene encoding 103P2D6 as a result of homologous recombination between the endogenous gene encoding 103P2D6 and altered genomic DNA encoding 103P2D6 introduced into an embryonic cell of the animal. For example, cDNA that encodes 103P2D6 can be used to clone genomic DNA encoding 103P2D6 in accordance with established techniques. A portion of the genomic DNA encoding 103P2D6 can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras (see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells. A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal, and the embryo brought to term to create a “knock out” animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knock out animals can be characterized, for example, for their ability to defend against certain pathological conditions or for their development of pathological conditions due to absence of the 103P2D6 polypeptide.

[0159] VIII.) Methods for the Detection of 103P2D6

[0160] Another aspect of the present invention relates to methods for detecting 103P2D6 polynucleotides and 103P2D6-related proteins, as well as methods for identifying a cell that expresses 103P2D6. The expression profile of 103P2D6 makes it a diagnostic marker for metastasized disease. Accordingly, the status of 103P2D6 gene products provides information useful for predicting a variety of factors including susceptibility to advanced stage disease, rate of progression, and/or tumor aggressiveness. As discussed in detail herein, the status of 103P2D6 gene products in patient samples can be analyzed by a variety protocols that are well known in the art including immunohistochemical analysis, the variety of Northern blotting techniques including in situ hybridization, RT-PCR analysis (for example on laser capture micro-dissected samples), Western blot analysis and tissue array analysis.

[0161] More particularly, the invention provides assays for the detection of 103P2D6 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable 103P2D6 polynucleotides include, for example, a 103P2D6 gene or fragment thereof, 103P2D6 mRNA, alternative splice variant 103P2D6 mRNAs, and recombinant DNA or RNA molecules that contain a 103P2D6 polynucleotide. A number of methods for amplifying and/or detecting the presence of 103P2D6 polynucleotides are well known in the art and can be employed in the practice of this aspect of the invention.

[0162] In one embodiment, a method for detecting an 103P2D6 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using an 103P2D6 polynucleotides as sense and antisense primers to amplify 103P2D6 cDNAs therein; and detecting the presence of the amplified 103P2D6 cDNA. Optionally, the sequence of the amplified 103P2D6 cDNA can be determined.

[0163] In another embodiment, a method of detecting a 103P2D6 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 103P2D6 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 103P2D6 gene. Any number of appropriate sense and antisense probe combinations can be designed from the nucleotide sequences provided for the 103P2D6 (FIG. 2) and used for this purpose.

[0164] The invention also provides assays for detecting the presence of an 103P2D6 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a 103P2D6-related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA and the like. For example, a method of detecting the presence of a 103P2D6-related protein in a biological sample comprises first contacting the sample with a 103P2D6 antibody, a 103P2D6-reactive fragment thereof, or a recombinant protein containing an antigen binding region of a 103P2D6 antibody; and then detecting the binding of 103P2D6-related protein in the sample.

[0165] Methods for identifying a cell that expresses 103P2D6 are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 103P2D6 gene comprises detecting the presence of 103P2D6 mRNA in the cell. Methods for the detection of particular mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled 103P2D6 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 103P2D6, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like). Alternatively, an assay for identifying a cell that expresses a 103P2D6 gene comprises detecting the presence of 103P2D6-related protein in the cell or secreted by the cell. Various methods for the detection of proteins are well known in the art and are employed for the detection of 103P2D6-related proteins and cells that express 103P2D6-related proteins.

[0166] 103P2D6 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 103P2D6 gene expression. For example, 103P2D6 expression is significantly upregulated in prostate cancer, and is expressed in cancers of the tissues listed in Table I. Identification of a molecule or biological agent that inhibits 103P2D6 expression or over-expression in cancer cells is of therapeutic value. For example, such an agent can be identified by using a screen that quantifies 103P2D6 expression by RT-PCR, nucleic acid hybridization or antibody binding.

[0167] VIII.) Methods for Monitoring the Status of 103P2D6-Related Genes and Their Products

[0168] Oncogenesis is known to be a multistep process where cellular growth becomes progressively dysregulated and cells progress from a normal physiological state to precancerous and then cancerous states (see, e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al., Cancer Surv. 23: 19-32 (1995)). In this context, examining a biological sample for evidence of dysregulated cell growth (such as aberrant 103P2D6 expression in cancers) allows for early detection of such aberrant physiology, before a pathologic state such as cancer has progressed to a stage that therapeutic options are more limited and or the prognosis is worse. In such examinations, the status of 103P2D6 in a biological sample of interest can be compared, for example, to the status of 103P2D6 in a corresponding normal sample (e.g. a sample from that individual or alternatively another individual that is not effected by a pathology). An alteration in the status of 103P2D6 in the biological sample (as compared to the normal sample) provides evidence of dysregulated cellular growth. In addition to using a biological sample that is not effected by a pathology as a normal sample, one can also use a predetermined normative value such as a predetermined normal level of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol. Dec. 9, 1996;376(2):306-14 and U.S. Pat. No. 5,837,501) to compare 103P2D6 status in a sample.

[0169] The term “status” in this context is used according to its art accepted meaning and refers to the condition or state of a gene and its products. Typically, skilled artisans use a number of parameters to evaluate the condition or state of a gene and its products. These include, but are not limited to the location of expressed gene products (including the location of 103P2D6 expressing cells) as well as the, level, and biological activity of expressed gene products (such as 103P2D6 mRNA polynucleotides and polypeptides). Typically, an alteration in the status of 103P2D6 comprises a change in the location of 103P2D6 and/or 103P2D6 expressing cells and/or an increase in 103P2D6 mRNA and/or protein expression.

[0170] 103P2D6 status in a sample can be analyzed by a number of means well known in the art, including without limitation, immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture micro-dissected samples, Western blot analysis, and tissue array analysis. Typical protocols for evaluating the status of the 103P2D6 gene and gene products are found, for example in Ausubul et al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the status of 103P2D6 in a biological sample is evaluated by various methods utilized by skilled artisans including, but not limited to genomic Southern analysis (to examine, for example perturbations in the 103P2D6 gene), Northern analysis and/or PCR analysis of 103P2D6 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 103P2D6 m-RNAs), and, Western and/or immunohistochemical analysis (to examine, for example alterations in polypeptide sequences, alterations in polypeptide localization within a sample, alterations in expression levels of 103P2D6 proteins and/or associations of 103P2D6 proteins with polypeptide binding partners). Detectable 103P2D6 polynucleotides include, for example, a 103P2D6 gene or fragment thereof, 103P2D6 mRNA, alternative splice variants, 103P2D6 mRNAs, and recombinant DNA or RNA molecules containing a 103P2D6 polynucleotide.

[0171] The expression profile of 103P2D6 makes it a diagnostic marker for local and/or metastasized disease, and provides information on the growth or oncogenic potential of a biological sample. In particular, the status of 103P2D6 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining 103P2D6 status and diagnosing cancers that express 103P2D6, such as cancers of the tissues listed in Table I. For example, because 103P2D6 mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of 103P2D6 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 103P2D6 dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.

[0172] The expression status of 103P2D6 provides information including the presence, stage and location of dysplastic, precancerous and cancerous cells, predicting susceptibility to various stages of disease, and/or for gauging tumor aggressiveness. Moreover, the expression profile makes it useful as an imaging reagent for metastasized disease. Consequently, an aspect of the invention is directed to the various molecular prognostic and diagnostic methods for examining the status of 103P2D6 in biological samples such as those from individuals suffering from, or suspected of suffering from a pathology characterized by dysregulated cellular growth, such as cancer.

[0173] As described above, the status of 103P2D6 in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 103P2D6 in a biological sample taken from a specific location in the body can be examined by evaluating the sample for the presence or absence of 103P2D6 expressing cells (e.g. those that express 103P2D6 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 103P2D6-expressing cells are found in a biological sample that does not normally contain such cells (such as a lymph node), because such alterations in the status of 103P2D6 in a biological sample are often associated with dysregulated cellular growth. Specifically, one indicator of dysregulated cellular growth is the metastases of cancer cells from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node). In this context, evidence of dysregulated cellular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, e.g., Murphy et al., Prostate 42(4): 315-317 (2000);Su et al., Sernin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol August 1995;154(2 Pt 1):474-8).

[0174] In one aspect, the invention provides methods for monitoring 103P2D6 gene products by determining the status of 103P2D6 gene products expressed by cells from an individual suspected of having a disease associated with dysregulated cell growth (such as hyperplasia or cancer) and then comparing the status so determined to the status of 103P2D6 gene products in a corresponding normal sample. The presence of aberrant 103P2D6 gene products in the test sample relative to the normal sample provides an indication of the presence of dysregulated cell growth within the cells of the individual.

[0175] In another aspect, the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase in 103P2D6 mRNA or protein expression in a test cell or tissue sample relative to expression levels in the corresponding normal cell or tissue. The presence of 103P2D6 mRNA can, for example, be evaluated in tissue samples including but not limited to those listed in Table I. The presence of significant 103P2D6 expression in any of these tissues is useful to indicate the emergence, presence and/or severity of a cancer, since the corresponding normal tissues do not express 103P2D6 mRNA or express it at lower levels.

[0176] In a related embodiment, 103P2D6 status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 103P2D6 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 103P2D6 expressed in a corresponding normal sample. In one embodiment, the presence of 103P2D6 protein is evaluated, for example, using immunohistochemical methods. 103P2D6 antibodies or binding partners capable of detecting 103P2D6 protein expression are used in a variety of assay formats well known in the art for this purpose.

[0177] In a further embodiment, one can evaluate the status 103P2D6 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules. These perturbations can include insertions, deletions, substitutions and the like. Such evaluations are useful because perturbations in the nucleotide and amino acid sequences are observed in a large number of proteins associated with a growth dysregulated phenotype (see, e.g., Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, a mutation in the sequence of 103P2D6 may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 103P2D6 indicates a potential loss of function or increase in tumor growth.

[0178] A wide variety of assays for observing perturbations in nucleotide and amino acid sequences are well known in the art. For example, the size and structure of nucleic acid or amino acid sequences of 103P2D6 gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods for observing perturbations in nucleotide and amino acid sequences such as single strand conformation polymorphism analysis are well known in the art (see, e.g., U.S. Pat. No. 5,382,510 issued Sep. 7, 1999, and U.S. Pat. No. 5,952,170 issued Jan. 17, 1995).

[0179] Additionally, one can examine the methylation status of the 103P2D6 gene in a biological sample. Aberrant demethylation and/or hypermethylation of CpG islands in gene 5′ regulatory regions frequently occurs in immortalized and transformed cells, and can result in altered expression of various genes. For example, promoter hypermethylation of the pi-class glutathione S-transferase (a protein expressed in normal prostate but not expressed in >90% of prostate carcinomas) appears to permanently silence transcription of this gene and is the most frequently detected genomic alteration in prostate carcinomas (De Marzo et al., Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration is present in at least 70% of cases of high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et al, Cancer Epidemiol. Biomarkers Prev., 1998, 7:531-536). In another example, expression of the LAGE-I tumor specific gene (which is not expressed in normal prostate but is expressed in 25-50% of prostate cancers) is induced by deoxy-azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). A variety of assays for examining methylation status of a gene are well known in the art. For example, one can utilize, in Southern hybridization approaches, methylation-sensitive restriction enzymes which cannot cleave sequences that contain methylated CpG sites to assess the methylation status of CpG islands. In addition, MSP (methylation specific PCR) can rapidly profile the methylation status of all the CpG sites present in a CpG island of a given gene. This procedure involves initial modification of DNA by sodium bisulfite (which will convert all unmethylated cytosines to uracil) followed by amplification using primers specific for methylated versus unmethylated DNA. Protocols involving methylation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubul et al. eds., 1995.

[0180] Gene amplification is an additional method for assessing the status of 103P2D6. Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies are employed that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn are labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

[0181] Biopsied tissue or peripheral blood can be conveniently assayed for the presence of cancer cells using for example, Northern, dot blot or RT-PCR analysis to detect 103P2D6 expression. The presence of RT-PCR amplifiable 103P2D6 mRNA provides an indication of the presence of cancer. RT-PCR assays are well known in the art. RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and management of a number of human solid tumors. In the prostate cancer field, these include RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000; Heston et al., 1995, Clin. Chem. 41:1687-1688).

[0182] A further aspect of the invention is an assessment of the susceptibility that an individual has for developing cancer. In one embodiment, a method for predicting susceptibility to cancer comprises detecting 103P2D6 mRNA or 103P2D6 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 103P2D6 mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 103P2D6 in prostate or other tissue is examined, with the presence of 103P2D6 in the sample providing an indication of prostate cancer susceptibility (or the emergence or existence of a prostate tumor). Similarly, one can evaluate the integrity 103P2D6 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations in 103P2D6 gene products in the sample is an indication of cancer susceptibility (or the emergence or existence of a tumor).

[0183] The invention also comprises methods for gauging tumor aggressiveness. In one embodiment, a method for gauging aggressiveness of a tumor comprises determining the level of 103P2D6 mRNA or 103P2D6 protein expressed by tumor cells, comparing the level so determined to the level of 103P2D6 mRNA or 103P2D6 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 103P2D6 mRNA or 103P2D6 protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness. In a specific embodiment, aggressiveness of a tumor is evaluated by determining the extent to which 103P2D6 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 103P2D6 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations indicates more aggressive tumors.

[0184] Another embodiment of the invention is directed to methods for observing the progression of a malignancy in an individual over time. In one embodiment, methods for observing the progression of a malignancy in an individual over time comprise determining the level of 103P2D6 mRNA or 103P2D6 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 103P2D6 mRNA or 103P2D6 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 103P2D6 mRNA or 103P2D6 protein expression in the tumor sample over time provides information on the progression of the cancer. In a specific embodiment, the progression of a cancer is evaluated by determining 103P2D6 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 103P2D6 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like, where the presence of one or more perturbations indicates a progression of the cancer.

[0185] The above diagnostic approaches can be combined with any one of a wide variety of prognostic and diagnostic protocols known in the art. For example, another embodiment of the invention is directed to methods for observing a coincidence between the expression of 103P2D6 gene and 103P2D6 gene products (or perturbations in 103P2D6 gene and 103P2D6 gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a tissue sample. A wide variety of factors associated with malignancy can be utilized, such as the expression of genes associated with malignancy (e.g. PSA, PSCA and PSM expression for prostate cancer etc.) as well as gross cytological observations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol. 6(2):74-88; Eptsein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al., 1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing a coincidence between the expression of 103P2D6 gene and 103P2D6 gene products (or perturbations in 103P2D6 gene and 103P2D6 gene products) and another factor that is associated with malignancy are useful, for example, because the presence of a set of specific factors that coincide with disease provides information crucial for diagnosing and prognosticating the status of a tissue sample.

[0186] In one embodiment, methods for observing a coincidence between the expression of 103P2D6 gene and 103P2D6 gene products (or perturbations in 103P2D6 gene and 103P2D6 gene products) and another factor associated with malignancy entails detecting the overexpression of 103P2D6 mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of 103P2D6 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of 103P2D6 and PSA mRNA in prostate tissue is examined, where the coincidence of 103P2D6 and PSA mRNA overexpression in the sample indicates the existence of prostate cancer, prostate cancer susceptibility or the emergence or status of a prostate tumor.

[0187] Methods for detecting and quantifying the expression of 103P2D6 mRNA or protein are described herein, and standard nucleic acid and protein detection and quantification technologies are well known in the art. Standard methods for the detection and quantification of 103P2D6 mRNA include in situ hybridization using labeled 103P2D6 riboprobes, Northern blot and related techniques using 103P2D6 polynucleotide probes, RT-PCR analysis using primers specific for 103P2D6, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantify 103P2D6 mRNA expression. Any number of primers capable of amplifying 103P2D6 can be used for this purpose, including but not limited to the various primer sets specifically described herein. In a specific embodiment, polyclonal or monoclonal antibodies specifically reactive with the wild-type 103P2D6 protein can be used in an immunohistochemical assay of biopsied tissue.

[0188] IX.) Identifying Molecules that Interact with 103P2D6

[0189] The 103P2D6 protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 103P2D6, as well as pathways activated by 103P2D6 via any one of a variety of art accepted protocols. For example, one can utilize one of the so-called interaction trap systems (also referred to as the “two-hybrid assay”). In such systems, molecules interact and reconstitute a transcription factor which directs expression of a reporter gene, whereupon the expression of the reporter gene is assayed. Other systems identify protein-protein interactions in vivo through reconstitution of a eukaryotic transcriptional activator, see, e.g., U.S. Pat. No. 5,955,280 issued Sep. 21, 1999, U.S. Pat. No. 5,925,523 issued Jul. 20, 1999, U.S. Pat. No. 5,846,722 issued Dec. 8, 1998 and U.S. Pat. No. 6,004,746 issued Dec. 21, 1999.

[0190] Alternatively one can screen peptide libraries to identify molecules that interact with 103P2D6 protein sequences. In such methods, peptides that bind to a molecule such as 103P2D6 are identified by screening libraries that encode a random or controlled collection of amino acids. Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, the bacteriophage particles are then screened against the protein of interest.

[0191] Accordingly, peptides having a wide variety of uses, such as therapeutic, prognostic or diagnostic reagents, are thus identified without any prior information on the structure of the expected ligand or receptor molecule. Typical peptide libraries and screening methods that can be used to identify molecules that interact with 103P2D6 protein sequences are disclosed for example in U.S. Pat. No. 5,723,286 issued Mar. 3, 1998 and U.S. Pat. No. 5,733,731 issued Mar. 31, 1998.

[0192] Alternatively, cell lines that express 103P2D6 are used to identify protein-protein interactions mediated by 103P2D6. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton B J, et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 103P2D6 protein can be immunoprecipitated from 103P2D6-expressing cell lines using anti-103P2D6 antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express 103P2D6 (vectors mentioned above). The immunoprecipitated complex can be examined for protein association by procedures such as Western blotting, ³⁵S-methionine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.

[0193] Small molecules and ligands that interact with 103P2D6 can be identified through related embodiments of such screening assays. For example, small molecules can be identified that interfere with protein function, including molecules that interfere with 103P2D6's ability to mediate phosphorylation and de-phosphorylation, second messenger signaling or tumorigenesis. Similarly, ligands that regulate 103P2D6 function can be identified based on their ability to bind 103P2D6 and activate a reporter construct. Typical methods are discussed for example in U.S. Pat. No. 5,928,868 issued Jul. 27, 1999, and include methods for forming hybrid ligands in which at least one ligand is a small molecule. In an illustrative embodiment, cells engineered to express a fusion protein of 103P2D6 and a DNA-binding protein are used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library transcriptional activator protein. The cells further contain a reporter gene, the expression of which is conditioned on the proximity of the first and second fusion proteins to each other, an event that occurs only if the hybrid ligand binds to target sites on both hybrid proteins. Those cells that express the reporter gene are selected and the unknown small molecule or the unknown ligand is identified. This method provides a means of identifying both activators and inhibitors of 103P2D6.

[0194] An embodiment of this invention comprises a method of screening for a molecule that interacts with an 103P2D6 amino acid sequence shown in FIG. 2 and FIG. 3, comprising the steps of contacting a population of molecules with the 103P2D6 amino acid sequence, allowing the population of molecules and the 103P2D6 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 103P2D6 amino acid sequence, and then separating molecules that do not interact with the 103P2D6 amino acid sequence from molecules that do. In a specific embodiment, the method further comprises purifying a molecule that interacts with the 103P2D6 amino acid sequence. The identified molecule can be used to modulate a function performed by 103P2D6. In a preferred embodiment, the 103P2D6 amino acid sequence is contacted with a library of peptides.

[0195] X.) Therapeutic Methods and Compositions

[0196] The identification of 103P2D6 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in prostate and other cancers, opens a number of therapeutic approaches to the treatment of such cancers. As discussed herein, it is possible that 103P2D6 functions as a transcription factor involved in activating tumor-promoting genes or repressing genes that block tumorigenesis.

[0197] Accordingly, therapeutic approaches that inhibit the activity of the 103P2D6 protein are useful for patients suffering a cancer that expresses 103P2D6. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of the 103P2D6 protein with its binding partner or with others proteins. Another class comprises a variety of methods for inhibiting the transcription of the 103P2D6 gene or translation of 103P2D6 mRNA.

[0198] X.A.) 103P2D6 as a Target for Antibody-Based Therapy

[0199] 103P2D6 is an attractive target for antibody-based therapeutic strategies. A number of antibody strategies are known in the art for targeting both extracellular and intracellular molecules (see, e.g., complement and ADCC mediated killing as well as the use of intrabodies). Because 103P2D6 is expressed by cancer cells of various lineages and not by corresponding normal cells, systemic administration of 103P2D6-immunoreactive compositions are prepared that exhibit excellent sensitivity without toxic, non-specific and/or non-target effects caused by binding of the immunoreactive composition to non-target organs and tissues. Antibodies specifically reactive with domains of 103P2D6 are useful to treat 103P2D6-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.

[0200] 103P2D6 antibodies can be introduced into a patient such that the antibody binds to 103P2D6 and modulates a function, such as an interaction with a binding partner, and consequently mediates destruction of the tumor cells and/or inhibits the growth of the tumor cells. Mechanisms by which such antibodies exert a therapeutic effect can include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity, modulation of the physiological function of 103P2D6, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of tumor angiogenesis factor profiles, and/or apoptosis.

[0201] Those skilled in the art understand that antibodies can be used to specifically target and bind immunogenic molecules such as an immunogenic region of the 103P2D6 sequence shown in FIG. 2. In addition, skilled artisans understand that it is routine to conjugate antibodies to cytotoxic agents. When cytotoxic and/or therapeutic agents are delivered directly to cells, such as by conjugating them to antibodies specific for a molecule expressed by that cell (e.g. 103P2D6), the cytotoxic agent will exert its known biological effect (i.e. cytotoxicity) on those cells.

[0202] A wide variety of compositions and methods for using antibody-cytotoxic agent conjugates to kill cells are known in the art. In the context of cancers, typical methods entail administering to an animal having a tumor a biologically effective amount of a conjugate comprising a selected cytotoxic and/or therapeutic agent linked to a targeting agent (e.g. an anti-103P2D6 antibody) that binds to a marker (e.g. 103P2D6) expressed, accessible to binding or localized on the cell surfaces. A typical embodiment is a method of delivering a cytotoxic and/or therapeutic agent to a cell expressing 103P2D6, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 103P2D6 epitope, and, exposing the cell to the antibody-agent conjugate. Another illustrative embodiment is a method of treating an individual suspected of suffering from metastasized cancer, comprising a step of administering parenterally to said individual a pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent.

[0203] Cancer immunotherapy using anti-103P2D6 antibodies can be done in accordance with various approaches that have been successfully employed in the treatment of other types of cancer, including but not limited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994, Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res. 55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin. Immunol. 11:117-127). Some therapeutic approaches involve conjugation of naked antibody to a toxin, such as the conjugation of ¹³¹I to anti-CD20 antibodies (e.g., Rituxan™, IDEC Pharmaceuticals Corp.), while others involve co-administration of antibodies and other therapeutic agents, such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). To treat prostate cancer, for example, 103P2D6 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation.

[0204] Although 103P2D6 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well.

[0205] Cancer patients can be evaluated for the presence and level of 103P2D6 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 103P2D6 imaging, or other techniques that reliably indicate the presence and degree of 103P2D6 expression. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissues are well known in the art.

[0206] Anti-103P2D6 monoclonal antibodies that treat prostate and other cancers include those that initiate a potent immune response against the tumor or those that are directly cytotoxic. In this regard, anti-103P2D6 monoclonal antibodies (mAbs) can elicit tumor cell lysis by either complement-mediated or antibody-dependent cell cytotoxicity (ADCC) mechanisms, both of which require an intact Fc portion of the immunoglobulin molecule for interaction with effector cell Fc receptor sites on complement proteins. In addition, anti-103P2D6 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 103P2D6. Mechanisms by which directly cytotoxic mAbs act include: inhibition of cell growth, modulation of cellular differentiation, modulation of tumor angiogenesis factor profiles, and the induction of apoptosis. The mechanism(s) by which a particular anti-103P2D6 mAb exerts an anti-tumor effect is evaluated using any number of in vitro assays that evaluate cell death such as ADCC, ADMMC, complement-mediated cell lysis, and so forth, as is generally known in the art.

[0207] In some patients, the use of murine or other non-human monoclonal antibodies, or human/mouse chimeric mAbs can induce moderate to strong immune responses against the non-human antibody. This can result in clearance of the antibody from circulation and reduced efficacy. In the most severe cases, such an immune response can lead to the extensive formation of immune complexes which, potentially, can cause renal failure. Accordingly, preferred monoclonal antibodies used in the therapeutic methods of the invention are those that are either fully human or humanized and that bind specifically to the target 103P2D6 antigen with high affinity but exhibit low or no antigenicity in the patient.

[0208] Therapeutic methods of the invention contemplate the administration of single anti-103P2D6 mAbs as well as combinations, or cocktails, of different mabs. Such mAb cocktails can have certain advantages inasmuch as they contain mAbs that target different epitopes, exploit different effector mechanisms or combine directly cytotoxic mAbs with mAbs that rely on immune effector functionality. Such mAbs in combination can exhibit synergistic therapeutic effects. In addition, anti-103P2D6 mAbs can be administered concomitantly with other therapeutic modalities, including but not limited to various chemotherapeutic agents, androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery or radiation. The anti-103P2D6 mAbs are administered in their “naked” or unconjugated form, or can have a therapeutic agent(s) conjugated to them.

[0209] Anti-103P2D6 antibody formulations are administered via any route capable of delivering the antibodies to a tumor cell. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like. Treatment generally involves repeated administration of the anti-103P2D6 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1 to about 10 mg/kg body weight. In general, doses in the range of 10-500 mg mAb per week are effective and well tolerated.

[0210] Based on clinical experience with the Herceptin mAb in the treatment of metastatic breast cancer, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-103P2D6 mAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose is administered as a 90 minute or longer infusion. The periodic maintenance dose is administered as a 30 minute or longer infusion, provided the initial dose was well tolerated. As appreciated by those of skill in the art, various factors can influence the ideal dose regimen in a particular case. Such factors include, for example, the binding affinity and half life of the Ab or mAbs used, the degree of 103P2D6 expression in the patient, the extent of circulating shed 103P2D6 antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient.

[0211] Optionally, patients should be evaluated for the levels of 103P2D6 in a given sample (e.g. the levels of circulating 103P2D6 antigen and/or 103P2D6 expressing cells) in order to assist in the determination of the most effective dosing regimen, etc. Such evaluations are also used for monitoring purposes throughout therapy, and are useful to gauge therapeutic success in combination with the evaluation of other parameters (such as serum PSA levels in prostate cancer therapy).

[0212] X.B.) Anti-Cancer Vaccines

[0213] The invention further provides cancer vaccines comprising a 103P2D6-related protein or 103P2D6-related nucleic acid. In view of the expression of 103P2D6, cancer vaccines prevent and/or treat 103P2D6-expressing cancers without creating non-specific effects on non-target tissues. The use of a tumor antigen in a vaccine that generates humoral and/or cell-mediated immune responses as anti-cancer therapy is well known in the art and has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al., 1997, J. Immunol. 159:3113-3117).

[0214] Genetic immunization methods can be employed to generate prophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing 103P2D6. Constructs comprising DNA encoding a 103P2D6-related protein/immunogen and appropriate regulatory sequences can be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded 103P2D6 protein/immunogen. Alternatively, a vaccine comprises a 103P2D6-related protein. Expression of the 103P2D6-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear 103P2D6 protein. Various prophylactic and therapeutic genetic immunization techniques known in the art can be used (for review, see information and references published at Internet address www.genweb.com).

[0215] Such methods can be readily practiced by employing a 103P2D6-related protein, or an 103P2D6-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 103P2D6 immunogen (which typically comprises a number of antibody or T cell epitopes). Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, e.g., Heryln et al., Ann Med February 1999;31(1):66-78; Maruyama et al., Cancer Immunol Immunother June 2000;49(3):123-32) Briefly, such methods of generating an immune response (e.g. humoral and/or cell-mediated) in a mammal, comprise the steps of: exposing the manmmal's immune system to an immunoreactive epitope (e.g. an epitope present in the 103P2D6 protein shown in SEQ ID NO: 2 or analog or homolog thereof) so that the mammal generates an immune response that is specific for that epitope (e.g. generates antibodies that specifically recognize that epitope). In a preferred method, the 103P2D6 immunogen contains a biological motif.

[0216] CTL epitopes can be determined using specific algorithms to identify peptides within 103P2D6 protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV (A) and Table IV (B); Epimer™ and Epimatrix™, Brown University (http://www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and, BIMAS, (http://bimas.dert.nih.gov/). In a preferred embodiment, the 103P2D6 immunogen contains one or more amino acid sequences identified using one of the pertinent analytical techniques well known in the art, such as the sequences shown in Tables V-XVIII or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I motif (e.g., Table IV (A)) and/or a peptide of at least 9 amino acids that comprises an HLA Class II motif (e.g., Table IV (B)). As is appreciated in the art, the HLA Class I binding grove is essentially closed ended so that peptides of only a particular size range can fit into the groove and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLA Class II binding groove is essentially open ended; therefore a of about 9 or more amino acids can be bound by an HLA Class II molecule. Due to the binding groove differences between HLA Class I and II, HLA Class I motifs are length specific, i.e., position two of a Class I motif is the second amino acid in an amino to carboxyl direction of the peptide. The amino acid positions in a Class II motif are relative only to each other, not the overall peptide, i.e., additional amino acids can be attached to the amino and/or carboxyl termini of a motif-bearing sequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long, but can be longer than 25 amino acids.

[0217] A wide variety of methods for generating an immune response in a mammal are known in the art (for example as the first step in the generation of hybridomas). Methods of generating an immune response in a mammal comprise exposing the mammal's immune system to an immunogenic epitope on a protein (e.g. the 103P2D6 protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 103P2D6 in a host, by contacting the host with a sufficient amount of at least one 103P2D6 B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 103P2D6 B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 103P2D6-related protein or a man-made multiepitopic peptide comprising: administering 103P2D6 immunogen (e.g. the 103P2D6 protein or a peptide fragment thereof, an 103P2D6 fusion protein or analog etc.) in a vaccine preparation to a human or another mammal. Typically, such vaccine preparations further contain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or a universal helper epitope such as a PADRE™ peptide (Epimmune Inc., San Diego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92). An alternative method comprises generating an immune response in an individual against a 103P2D6 immunogen by: administering in vivo to muscle or skin of the individual's body a DNA molecule that comprises a DNA sequence that encodes an 103P2D6 immunogen, the DNA sequence operatively linked to regulatory sequences which control the expression of the DNA sequence; wherein the DNA molecule is taken up by cells, the DNA sequence is expressed in the cells and an immune response is generated against the immunogen (see, e.g., U.S. Pat. No. 5,962,428). The DNA can be dissociated from an infectious agent. Optionally a genetic vaccine facilitator such as anionic lipids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea is also administered.

[0218] Thus, viral gene delivery systems are used to deliver a 103P2D6-related nucleic acid molecule. Various viral gene delivery systems that can be used in the practice of the invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus, and sindbus virus (Restifo, 1996, Curr. Opin. Immunol. 8:658-663). Non-viral delivery systems can also be employed by introducing naked DNA encoding a 103P2D6-related protein into the patient (e.g., intramuscularly or intradermally) to induce an anti-tumor response. In one embodiment, the full-length human 103P2D6 cDNA is employed. In another embodiment, 103P2D6 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopes are employed.

[0219] Various ex vivo strategies can also be employed to generate an immune response. One approach involves the use of antigen presenting cells (APCs) such as dendritic cells to present 103P2D6 antigen to a patient's immune system. Dendritic cells express MHC class I and II molecules, B7 co-stimulator, and IL-12, and are thus highly specialized antigen presenting cells. In prostate cancer, autologous dendritic cells pulsed with peptides of the prostate-specific membrane antigen (PSMA) are being used in a Phase I clinical trial to stimulate prostate cancer patients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphy et al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used to present 103P2D6 peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 103P2D6 peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 103P2D6 protein. Yet another embodiment involves engineering the overexpression of the 103P2D6 gene in dendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur et al., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNA transfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), or tumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med. 186:1177-1182). Cells that express 103P2D6 can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.

[0220] Anti-idiotypic anti-103P2D6 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 103P2D6-related protein. In particular, the generation of anti-idiotypic antibodies is well known in the art; this methodology can readily be adapted to generate anti-idiotypic anti-103P2D6 antibodies that mimic an epitope on a 103P2D6-related protein (see, for example, Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin. Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccine strategies.

[0221] XI.) Inhibition of 103P2D6 Protein Function

[0222] The invention includes various methods and compositions for inhibiting the binding of 103P2D6 to its binding partner or its association with other protein(s) as well as methods for inhibiting 103P2D6 function.

[0223] XI.A.) Inhibition of 103P2D6 with Intracellular Antibodies

[0224] In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 103P2D6 are introduced into 103P2D6 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-103P2D6 antibody is expressed intracellularly, binds to 103P2D6 protein, and thereby inhibits its function. Methods for engineering such intracellular single chain antibodies are well known. Such intracellular antibodies, also known as “intrabodies”, are specifically targeted to a particular compartment within the cell, providing control over where the inhibitory activity of the treatment is focused. This technology has been successfully applied in the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors (see, e.g., Richardson et al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).

[0225] Single chain antibodies comprise the variable domains of the heavy and light chain joined by a flexible linker polypeptide, and are expressed as a single polypeptide. Optionally, single chain antibodies are expressed as a single chain variable region fragment joined to the light chain constant region. Well-known intracellular trafficking signals are engineered into recombinant polynucleotide vectors encoding such single chain antibodies in order to precisely target the intrabody to the desired intracellular compartment. For example, intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal, such as the KDEL amino acid motif. Intrabodies intended to exert activity in the nucleus are engineered to include a nuclear localization signal. Lipid moieties are joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert function in the cytosol. For example, cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.

[0226] In one embodiment, intrabodies are used to capture 103P2D6 in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such 103P2D6 intrabodies in order to achieve the desired targeting. Such 103P2D6 intrabodies are designed to bind specifically to a particular 103P2D6 domain. In another embodiment, cytosolic intrabodies that specifically bind to the 103P2D6 protein are used to prevent 103P2D6 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing 103P2D6 from forming transcription complexes with other factors).

[0227] In order to specifically direct the expression of such intrabodies to particular cells, the transcription of the intrabody is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer. In order to target intrabody expression specifically to prostate, for example, the PSA promoter and/or promoter/enhancer can be utilized (See, for example, U.S. Pat. No. 5,919,652 issued Jul. 6, 1999).

[0228] XI.B.) Inhibition of 103P2D6 with Recombinant Proteins

[0229] In another approach, recombinant molecules bind to 103P2D6 and thereby inhibit 103P2D6 function. For example, these recombinant molecules prevent or inhibit 103P2D6 from accessing/binding to its binding partner(s) or associating with other protein(s). Such recombinant molecules can, for example, contain the reactive part(s) of a 103P2D6 specific antibody molecule. In a particular embodiment the 103P2D6 binding domain of a 103P2D6 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 103P2D6 ligand binding domains linked to the Fc portion of a human IgG, such as human IgG1. Such IgG portion can contain, for example, the C_(H)2 and C_(H)3 domains and the hinge region, but not the C_(H)1 domain. Such dimeric fusion proteins are administered in soluble form to patients suffering from a cancer associated with the expression of 103P2D6, whereby the dimeric fusion protein specifically binds to 103P2D6 and blocks 103P2D6 interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.

[0230] XI.C.) Inhibition of 103P2D6 Transcription or Translation

[0231] The present invention also comprises various methods and compositions for inhibiting the transcription of the 103P2D6 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 103P2D6 mRNA into protein.

[0232] In one approach, a method of inhibiting the transcription of the 103P2D6 gene comprises contacting the 103P2D6 gene with a 103P2D6 antisense polynucleotide. In another approach, a method of inhibiting 103P2D6 mRNA translation comprises contacting the 103P2D6 mRNA with an antisense polynucleotide. In another approach, a 103P2D6 specific ribozyme is used to cleave the 103P2D6 message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 103P2D6 gene, such as the 103P2D6 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 103P2D6 gene transcription factor are used to inhibit 103P2D6 mRNA transcription. The various polynucleotides and compositions useful in the aforementioned methods have been described above. The use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.

[0233] Other factors that inhibit the transcription of 103P2D6 by interfering with 103P2D6 transcriptional activation are also useful to treat cancers expressing 103P2D6. Similarly, factors that interfere with 103P2D6 processing are useful to treat cancers that express 103P2D6. Cancer treatment methods utilizing such factors are also within the scope of the invention.

[0234] XI.D.) General Considerations for Therapeutic Strategies

[0235] Gene transfer and gene therapy technologies can be used to deliver therapeutic polynucleotide molecules to tumor cells synthesizing 103P2D6 (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 103P2D6 inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding 103P2D6 antisense polynucleotides, ribozymes, factors capable of interfering with 103P2D6 transcription, and so fort, can be delivered to target tumor cells using such gene therapy approaches.

[0236] The above therapeutic approaches can be combined with any one of a wide variety of surgical, chemotherapy or radiation therapy regimens. The therapeutic approaches of the invention can enable the use of reduced dosages of chemotherapy (or other therapies) and/or less frequent administration, an advantage for all patients and particularly for those that do not tolerate the toxicity of the chemotherapeutic agent well.

[0237] The anti-tumor activity of a particular composition (e.g., antisense, ribozyme, intrabody), or a combination of such compositions, can be evaluated using various in vitro and in vivo assay systems. In vitro assays that evaluate therapeutic activity include cell growth assays, soft agar assays and other assays indicative of tumor promoting activity, binding assays capable of determining the extent to which a therapeutic composition will inhibit the binding of 103P2D6 to a binding partner, etc.

[0238] In vivo, the effect of a 103P2D6 therapeutic composition can be evaluated in a suitable animal model. For example, xenogenic prostate cancer models can be used, wherein human prostate cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine 3: 402-408). For example, PCT Patent Application WO98/16628, Sawyers et al., published Apr. 23, 1998, describes various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of late stage disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like. In vivo assays that evaluate the promotion of apoptosis are useful in evaluating therapeutic compositions. In one embodiment, xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing nice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.

[0239] The therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed., 1980).

[0240] Therapeutic formulations can be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like. A preferred formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium Chloride for Injection, USP. Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing for example, benzyl alcohol preservative) or in sterile water prior to injection.

[0241] Dosages and administration protocols for the treatment of cancers using the foregoing methods will vary with the method and the target cancer, and will generally depend on a number of other factors appreciated in the art.

[0242] XII.) Kits

[0243] For use in the diagnostic and therapeutic applications described herein, kits are also within the scope of the invention. Such kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method. For example, the container(s) can comprise a probe that is or can be detectably labeled. Such probe can be an antibody or polynucleotide specific for a 103P2D6-related protein or a 103P2D6 gene or message, respectively. Where the method utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label. The kit can include all or part of the amino acid sequence of FIG. 2 or analogs thereof, or a nucleic acid molecule that encodes such amino acid sequences.

[0244] The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0245] A label can be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic application, and can also indicate directions for either in vivo or in vitro use, such as those described above. Directions and or other information can also be included on an insert which is included with the kit.

[0246] p103P2D6-B (clone B) has been deposited under the requirements of the Budapest Treaty on May 19, 2000 with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209 USA, and has been identified as ATCC Accession No. PTA-1895. p 103P2D6-2 (clone 2) has been deposited under the requirements of the Budapest Treaty on Jan. 6, 2000 with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209 USA, and has been identified as ATCC Accession No. PTA-1155.

EXAMPLES

[0247] Various aspects of the invention are further described and illustrated by way of the several examples that follow, none of which are intended to limit the scope of the invention.

Example 1 SSH-Generated Isolation of a cDNA Fragment of the 103P2D6 Gene

[0248] Characterization of 103P2D6 by Suppression Subtractive Hybridization (SSH)

[0249] As discussed in detail below, experiments with the LAPC-4 AD xenograft in male SCID mice have resulted in the identification of genes that are involved in the progression of androgen dependent (AD) prostate cancer to androgen independent (Al) cancer. Briefly, mice that harbored LAPC-4 AD xenografts were castrated when the tumors reached a size of 1 cm in diameter. The tumors regressed in size and temporarily stopped producing the androgen dependent protein PSA. Seven to fourteen days post-castration, PSA levels were detectable again in the blood of the mice. Eventually such tumors develop an AI phenotype and start growing again in the castrated males. Tumors were harvested at different time points after castration to identify genes that are turned on or off during the transition to androgen independence.

[0250] Suppression subtractive hybridization (SSH) (Diatchenko et al., 1996, PNAS 93:6025) was then used to identify novel genes, such as those that are overexpressed in prostate cancer, by comparing cDNAs from various androgen dependent and androgen independent LAPC xenografts. This strategy resulted in the identification of novel genes exhibiting prostate cancer specific expression. One of these genes, designated 103P2D6, was identified from a subtraction where cDNA derived from an LAPC-4 AD tumor grown in an intact male was subtracted from cDNA derived from an LAPC-4 AD tumor, 21 days post-castration. The SSH DNA sequence of about 342 bp (FIG. 1) is novel and exhibits no homology to sequences in the public databases.

[0251] 103P2D6, encodes a transmembrane protein that exhibits tumor-specific expression. Expression analysis of 103P2D6 indicates that it is exclusively expressed in cancer of the prostate and other cancer tissues. 103P2D6 is expressed in fetal heart, kidney and lung, but not in normal adult tissues. The expression of 103P2D6 in cancer provides evidence that this protein has a functional role in tumor progression and/or initiation. It is possible that 103P2D6 functions as a receptor involved in activating or modulating proliferation signals involved in tumorigenesis and regulation of cell growth.

[0252] As is further described herein, the 103P2D6 gene and protein have been characterized using a number of analytical approaches. For example, analyses of nucleotide coding and amino acid sequences were conducted in order to identify potentially related molecules, as well as recognizable structural domains, topological features, and other elements within the 103P2D6 mRNA and protein structures. Northern blot analyses of 103P2D6 mRNA expression were conducted in order to establish the range of normal and cancerous tissues expressing 103P2D6 message.

[0253] Materials and Methods

[0254] LAPC Xenoyrafts and Human Tissues:

[0255] LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) and generated as described (Klein et al, 1997, Nature Med. 3: 402-408; Craft et al., 1999, Cancer Res. 59: 5030-5036). Androgen dependent and independent LAPC-4 xenografts (LAPC-4 AD and AI, respectively) and LAPC-9 xenografts (LAPC-9 AD and AI, respectively) were grown in intact male SCID mice or in castrated males, respectively, and were passaged as small tissue chunks in recipient males. LAPC-4 Al xenografts were derived from LAPC-4 AD tumors and LAPC-9 Al xenografts were derived from LAPC-9 AD tumors. To generate the AI xenografts, male mice bearing LAPC AD tumors were castrated and maintained for 2-3 months. After the LAPC tumors re-grew, the tumors were harvested and passaged in castrated males or in female SCID mice.

[0256] Cell Lines:

[0257] Human cell lines (e.g., HeLa) were obtained from the ATCC and were maintained in DMEM with 5% fetal calf serum.

[0258] RNA Isolation:

[0259] Tumor tissue and cell lines were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cells to isolate total RNA. Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Total and mRNA were quantified by spectrophotometric analysis (O.D. 260/280 nm) and analyzed by gel electrophoresis.

[0260] Oligonucleotides:

[0261] The following HPLC purified oligonucleotides were used.

[0262] DPNCDN (cDNA Synthesis Primer):

[0263] 5′TTTTGATCAAGCTT₃₀3′ (SEQ ID NO: 7) Adaptor 1: 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ ID NO: 8)                               3′CGGCCCGTCCTAG5′ (SEQ ID NO: 9) Adaptor 2: 5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO: 10)                               3′CGGCTCCTAG5′ (SEQ ID NO: 11) PCR primer 1: 5′CTAATACGACTCACTATAGGGC3′ (SEQ ID NO: 12) Nested primer (NP) 1: 5′TCGAGCGGCCGCCCGGGCAGGA3′ (SEQ ID NO: 13) Nested primer (NP)2: 5′AGCGTGGTCGCGGCCGAGGA3′ (SEQ ID NO: 14)

[0264] Suppression Subtractive Hybridization:

[0265] Suppression subtractive hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentially expressed in prostate cancer. The SSH reaction utilized cDNA from two LAPC-4 AD xenografts. Specifically, the 103P2D6 SSH sequence was identified from a subtraction where cDNA derived from an LAPC-4 AD tumor grown in an intact male was subtracted from cDNA derived from an LAPC-4 AD tumor, 21 days post-castration. The LAPC-4 AD xenograft tumor, 21 days post-castration, was used as the source of the “tester” cDNA, while the cDNA from the LAPC-4 AD tumor grown in an intact male was used as the source of the “driver” cDNA.

[0266] Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 μg of poly(A)⁺ RNA isolated from the relevant xenograft tissue, as described above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- and second-strand synthesis were carried out as described in the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3 hrs. at 37° C. Digested cDNA was extracted with phenol/chloroform (1:1) and ethanol precipitated.

[0267] Driver cDNA was generated by combining in a 1:1 ratio Dpn II digested cDNA from the relevant xenograft source (see above) with a mix of digested cDNAs derived from human benign prostatic hyperplasia (BPH), the human cell lines HeLa, 293, A431, Colo205, and mouse liver.

[0268] Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA from the relevant xenograft source (see above) (400 ng) in 5 μl of water. The diluted cDNA (2 μl, 160 ng) was then ligated to 2 II of Adaptor 1 and Adaptor 2 (10 μM), in separate ligation reactions, in a total volume of 10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C. for 5 min.

[0269] The first hybridization was performed by adding 1.5 III (600 ng) of driver cDNA to each of two tubes containing 1.5 μL (20 ng) Adaptor 1- and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, the samples were overlaid with mineral oil, denatured in an MJ Research thermal cycler at 98° C. for 1.5 minutes, and then were allowed to hybridize for 8 hrs at 68° C. The two hybridizations were then mixed together with an additional 1 μl of fresh denatured driver cDNA and were allowed to hybridize overnight at 68° C. The second hybridization was then diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA, heated at 70° C. for 7 min. and stored at −20° C.

[0270] PCR Amplification, Cloning and Sequencing of Gene Fragments Generated from SSH:

[0271] To amplify gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR reaction 1 μl of the diluted final hybridization mix was added to 1 μl of PCR primer 1 (10 μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10×reaction buffer (CLONTECH) and 0.5 μl 50×Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5 min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C. for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions were performed for each experiment. The products were pooled and diluted 1:10 with water. For the secondary PCR reaction, 1 μl from the pooled and diluted primary PCR reaction was added to the same reaction mix as used for PCR 1, except that primers NP1 and NP2 (10 μM) were used instead of PCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10 sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR products were analyzed using 2% agarose gel electrophoresis.

[0272] The PCR products were inserted into pCR2.1 using the T/A vector cloning kit (Invitrogen). Transformed E. coli were subjected to blue/white and ampicillin selection. White colonies were picked and arrayed into 96 well plates and were grown in liquid culture overnight. To identify inserts, PCR amplification was performed on 1 ml of bacterial culture using the conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose gel electrophoresis.

[0273] Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid DNA was prepared, sequenced, and subjected to nucleic acid homology searches of the GenBank, dbest, and NCI-CGAP databases.

[0274] Results

[0275] Two SSH experiments described in the Materials and Methods, supra, led to the isolation of numerous candidate gene fragment clones (SSH clones). All candidate clones were sequenced and subjected to homology analysis against all sequences in the major public gene and EST databases in order to provide information on the identity of the corresponding gene and to help guide the decision to analyze a particular gene for differential expression. In general, gene fragments that had no homology to any known sequence in any of the searched databases, and thus considered to represent novel genes, as well as gene fragments showing homology to previously sequenced expressed sequence tags (ESTs), were subjected to differential expression analysis by RT-PCR and/or northern analysis.

[0276] One of the SHH clones comprising about 342 bp, showed no homology to any known gene or to any sequences in the public databases, and was designated 103P2D6. Northern expression analysis of first strand cDNAs from 16 normal tissues showed a highly prostate tumor-specific expression pattern in adult tissues (FIG. 4).

Example 2 Full Length Cloning of 103P2D6 and Homology Comparison to Known Sequences

[0277] A partial 103P2D6 cDNA clone (clone 2) of 1687 base pairs (bp) was cloned from an LAPC-4 AD cDNA library (Lambda ZAP Express, Stratagene). A full-length 103P2D6 cDNA clone (FIG. 2) (clone B) of 4728 base pairs (b.p.) was cloned from a human fetal brain library (Pangene Inc.). The cDNA encodes a putative open reading frame (ORF) of 563 amino acids. Its calculated molecular weight (MW) is 63.4 kDa and its pI is 8.15. At the protein level, 103P2D6 shows 24.9% identity and 32.8% homology, taking account of any gaps, to an Envelope protein (Q9UNM3) isolated from a human endogenous retroviral protein HERV-H (Virology 1999, 258:441). The 103P2D6 nucleic acid sequence overlaps with some ESTs derived from kidney, fetus, brain and placenta.

[0278] The full-length 103P2D6 cDNA (clone B) was deposited on May 19, 2000, with the American Type Culture Collection (ATCC; Manassas, Va.) as plasmid p103P2D6-B, and has been assigned Accession No. PTA-1895. The partial 103P2D6 cDNA (clone 2) was deposited on Jan. 6, 2000, with the American Type Culture Collection (ATCC; Manassas, Va.) as plasmid p103P2D6-2, and has been assigned Accession No. PTA-1155.

Example 3 103P2D6 Gene & Protein Expression Analysis

[0279] Northern Expression Analysis:

[0280] 103P2D6 mRNA expression in normal human tissues was analyzed by northern blotting of multiple tissue blots (Clontech; Palo Alto, Calif.), comprising a total of 16 different normal human tissues, using labeled 103P2D6 SSH fragment (Example 1) as a probe. RNA samples were quantitatively normalized with a β-actin probe. Northern blot analysis using an 103P2D6 SSH fragment probe performed on 16 normal tissues failed to show expression in any normal tissues, including brain, ovary, heart, lung, liver, kidney, pancreas, small intestine, placenta, leukocytes, testis, prostate, colon, skeletal muscle, thymus and spleen (FIG. 5).

[0281] To analyze 103P2D6 expression in cancer tissues, northern blotting was performed on RNA derived from the LAPC xenografts, and several prostate and non-prostate cancer cell lines. The results show high expression levels of a 3 kb transcript in LAPC-4 AD, LAPC-4 Al, LAPC-9 AD, and LAPC-9 AI (FIG. 5, FIG. 6). More detailed analysis of the LAPC-4 xenografts shows that 103P2D6 is expressed at equal levels whether the xenografts are grown subcutaneously (LAPC-4 sc) or within the tibia of mice (LAPC-4 AD it) (FIG. 7). Expression was also detected in a xenograft that was grown within human bone explants in SCID mice (the LAPC-4 AD²). This indicates that bone growth of these prostate cancer tissues does not diminish their expression.

[0282] Expression of 103P2D6 was detected in several cancer cell lines derived from prostate (LNCaP, DU145, LAPC-4 CL), bladder (HT1197, 5637), pancreas (BxPC-3, HPAC, CAPAN-1), colon (SK-CO-1, CACO-2), bone (HOS, U2-OS, RD-ES), lung (CALU-1), breast (MCF-7), testis (NCCIT, TERA-1), cervix (A431), and ovary (OV-1063, PA-1, SW626) (FIG. 6). These results suggest that the 103P2D6 is generally up-regulated in cancer cells and cancer tissues, especially in prostate, kidney and bladder cancer, and serves as a suitable target for cancer therapy.

[0283] Northern blot analysis of 103P2D6 showed expression only in cancer tissues and cell lines, but not in normal adult tissues. Furthermore, the 103P2D6 gene was cloned from a fetal brain library, suggesting that it is a fetal gene that is activated during tumorigenesis. To investigate this hypothesis, 9 week old fetal RNA derived from 6 organs and from the whole embryo were analyzed by Northern blotting using a 103P2D6 SSH probe. The results (FIG. 8) show that 103P2D6 is expressed in fetal heart, kidney and lung at the 9 week stage. This indicates that 103P2D6 is indeed a fetal gene that is turned on in cancer and serves a role in cancer biology.

[0284] Expression of 103P2D6 was assayed in a panel of human cancers (T) and their respective matched normal tissues (N) on RNA dot blots (FIG. 13). 103P2D6 expression was seen in cancers of the kidney, breast, prostate, uterus, ovary, cervix, colon, stomach and rectum. 103P2D6 was also found to be highly expressed in the two human cancer cell lines, the CML line K562 and the colorectal carcinoma SW480. The expression detected in normal adjacent tissues (isolated from diseased tissues) but not in normal tissues, isolated from healthy donors, indicates that these tissues are not fully normal and that 103P2D6 may be expressed in early stage tumors and that it has utility as a diagnostic marker.

[0285]FIG. 14 shows Northern data where RNA was isolated from prostate tumors (T) and their adjacent normal tissues (N) obtained from the following prostate cancer patients (Pt); patient 1, Gleason score 4+5; patient 2, Gleason score 3+4; and, patient 3, Gleason score 4+3. Northern analysis was performed using 10 μg of total RNA for each sample. Expression of 103P2D6 was seen in all three tumor samples tested and their respective normal tissues.

[0286] Data from a Northern analysis where RNA was isolated from kidney tumors (T) and their adjacent normal tissues (N) obtained from kidney cancer patients is shown in FIG. 15. The patient specifications are as follows: Patient 1—Papillary Type, Stage I, Grade 2/4; Patient 2—Invasive papillary carcinoma, Grade 2/4; Patient 3—Clear cell type Grade 1/3, focally 2/3; Patient 4—Clear cell type, stage III, Grade 2/4; Patient 5—Clear cell type, stage III, Grade 3/4; Patient 6—Clear cell type, stage III, Grade 3/4; Patient 7—Clear cell type, Grade III. In FIG. 15, CL=Cell lines (from left to right): 769-P, A498, SW839; NK=Normal kidney; N=Normal adjacent tissue; T=Tumor. The Northern analysis was performed using 10 μg of total RNA for each sample. Elevated expression of 103P2D6 was observed in kidney tumors and normal adjacent tissues isolated from kidney cancer patients as compared to normal kidney.

[0287]FIG. 16 shows the results of Northern analysis where RNA was isolated from bladder cancers and adjacent normal tissue obtained from the bladder cancer patients. The Northern analysis was performed using 10 μg of total RNA for each sample. Expression of 103P2D6 was seen in bladder tumor but not in normal adjacent tissue.

[0288] RT-PCR Expression Analysis:

[0289] First strand cDNAs can be generated from 1 μg of mRNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplification system. The manufacturer's protocol was used, which included an incubation for 50 min at 42° C. with reverse transcriptase, followed by RNAse H treatment at 37° C. for 20 min. After completing the reaction, the volume can be increased to 200 μl with water prior to normalization. First strand cDNAs from 16 different normal human tissues can be obtained from Clontech.

[0290] Normalization of the first strand cDNAs from multiple tissues was performed by using the primers 5′-ATATCGCCGCGCTCGTCGTCGACAA-3′ (SEQ ID NO: XX) and 5′-AGCCACACGCAGCTCATTGTAGAAGG-3′ (SEQ ID NO: XX) to amplify β-actin. First strand cDNA (5 μl) were amplified in a total volume of 50 μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1XPCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH 8.3) and 1×Klentaq DNA polymerase (Clontech). Five μl of the PCR reaction can be removed at 18, 20, and 22 cycles and used for agarose gel electrophoresis. PCR was performed using an MJ Research thermal cycler under the following conditions: Initial denaturation can be at 94° C. for 15 sec, followed by a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C. for 5 sec. A final extension at 72° C. was carried out for 2 min. After agarose gel electrophoresis, the band intensities of the 283 b.p. β-actin bands from multiple tissues were compared by visual inspection. Dilution factors for the first strand cDNAs were calculated to result in equal β-actin band intensities in all tissues after 22 cycles of PCR. Three rounds of normalization can be required to achieve equal band intensities in all tissues after 22 cycles of PCR.

[0291] To determine expression levels of the 103P2D6 gene, 5 μl of normalized first strand cDNA were analyzed by PCR using 25, 30, and 35 cycles of amplification. Semi quantitative expression analysis can be achieved by comparing the PCR products at cycle numbers that give light band intensities.

[0292] In a typical RT-PCR expression analysis shown in FIG. 9, RT-PCR expression analysis was performed on first strand cDNAs generated using pools of tissues from multiple samples. The cDNAs were subsequently normalized using beta-actin PCR. The highest expression was observed in colon cancer pool, xenograft pool, and a lung cancer patient. Lower levels of expression were also observed normal prostate, prostate cancer, bladder cancer, and kidney cancer tissue pools.

[0293] In an additional analysis, RT-PCR was used to analyze expression of 103P2D6 in normal tissues and in patient-derived cancers. First strand cDNAs were generated from 1 μg of mRNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplification system. The manufacturers protocol was used and included an incubation for 50 min at 42° C. with reverse transcriptase followed by RNAse H treatment at 37° C. for 20 min. After completing the reaction, the volume was increased to 200 μl with water prior to normalization. First strand cDNAs were prepared from normal prostate, normal kidney and HeLa cancer cells, as well as a prostate tumor pool, a kidney tumor pool and a bladder tumor pool. The tumor pools were prepared from patient-derived tumor tissue. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR was performed using primers to 103P2D6. The 103P2D6 primers used for RT-PCR were: CTTGGGAGGTCCTAGTGCTAAGTG (SEQ. ID NO:) and CAATGAAGGGACTAACAACCCATC (SEQ. ID NO: XX). The results are shown in FIG. 9, and confirm that 103P2D6 is expressed in cancerous tissues, particularly in prostate and bladder cancer tissues derived from patients.

[0294] Protein Expression Analysis:

[0295] Expression of 103P2D6 protein was analyzed in pancreatic, colon, and prostate cancer cell lines using both western blot and flow cytometric analysis. As shown in FIGS. 11A-B, cell lysates (˜25 μg) from the indicated cell lines were separated by SDS-PAGE and subjected to Western blot analysis using an anti-103P2D6 pAb (see Example 4, below). Indicated with an arrow is a strong anti-103P2D6 pAb immunoreactive band of approximately 60 kD present in the pancreatic cancer cell lines HPAC and Bx PC-3, the colon cancer cell line CaCo-2, and a less intense band in LAPC9 prostate cancer cells, indicative of endogenous 103P2D6 protein expression. Also indicated with an arrow is the 85 kD immunoreactive band present in 293T cells transfected with V5-His tagged 103P2D6 cDNA.

[0296] Bx PC-3 pancreatic cancer cells were stained with anti-103P2D6 pAb (10 μg/ml) or control rabbit IgG Ab and subjected to flow cytometric analysis following incubation with anti-rabbit IgG-FITC conjugated secondary Ab. Bx PC-3 cells stained with the anti-103P2D6 pAb exhibited a fluorescence shift compared to the cells stained with control rabbit IgG, indicating cell surface expression of 103P2D6. The results are shown in FIG. 11C.

Example 4 Generation of 103P2D6 Polyclonal Antibodies

[0297] Generation of Polyclonal Antibodies (pAbs)

[0298] To generate polyclonal antibodies (pAb) to 103P2D6, a peptide was synthesized corresponding to amino acids 163-176 (DVTNESRNDDDDTS) of the 103P2D6 protein sequence. The peptide was coupled to Keyhole limpet hemacyanin (KLH) and used to immunize a rabbit as follows. The rabbit was initially immunized with 200 μg of peptide-KLH mixed in complete Freund's adjuvant. The rabbit was then injected every two to three weeks with 200 μg of peptide-KLH in incomplete Freund's adjuvant. Bleeds were taken approximately 7-10 days following each immunization. The titer of the serum was at least 1:64,000 as determined by ELISA to the peptide.

[0299] Affinity purified 103P2D6 polyclonal antibodies were prepared by passage of crude serum from the immunized rabbit over an affinity matrix comprised of 103P2D6 peptide covalently coupled to Affigel 10 (BioRad). After extensive washing of the matrix with PBS, antibodies specific to 103P2D6 peptide were eluted with low pH glycine buffer (0.1M, pH 2.5). Western blotting using the affinity purified pAb revealed the appearance of an anti-103P2D6 immunoreactive band of approximately 85 kD in 293T cells transiently transfected with the 103P2D6 cDNA in the pcDNA 3.1 V5-His vector, but not with the control empty vector (FIG. 12A). This pAb also detected cell surface expression of 103P2D6 protein in transfected 293T cells as determined by flow cytometry (FIG. 12B).

[0300] PAbs are also prepared by immunization of mice, rabbits, goats, and sheep with recombinant bacterial fusion proteins encoding full length or various regions of the 103P2D6 sequence. The recombinant bacterial proteins include glutathione-S-transferase (GST), maltose binding protein (MBP), and HIS tagged fusion proteins of 103P2D6 that are purified from induced bacteria using the appropriate affinity matrix. Mammalian expressed secreted Tag5 or FC-fusion proteins encoding the extracellular domain are also used as immunogens for pAb production.

[0301] Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. For example, 103P2D6, recombinant bacterial fusion proteins or peptides encoding various regions of the 103P2D6 sequence are used to immunize New Zealand White rabbits. Typically a peptide can be designed from a coding region of 103P2D6. The peptide can be conjugated to keyhole limpet hemocyanin (KLH) and used to immunize a rabbit. Alternatively the immunizing agent may include all or portions of the 103P2D6 protein, analogs or fusion proteins thereof. For example, the 103P2D6 amino acid sequence can be fused to any one of a variety of fusion protein partners that are well known in the art, such as maltose binding protein, LacZ, thioredoxin or an immunoglobulin constant region (see e.g. Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P.S., Brady, W., Umes, M., Grosmaire, L., Damle, N., and Ledbetter, L.(1991) J. Exp. Med. 174, 561-566). Other recombinant bacterial proteins include glutathione-S-transferase (GST), and HIS tagged fusion proteins of 103P2D6 that are purified from induced bacteria using the appropriate affinity matrix.

[0302] It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

[0303] In a typical protocol, rabbits are initially immunized subcutaneously with about 200 μg of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant. Rabbits are then injected subcutaneously every two weeks with 200 μg of immunogen in incomplete Freund's adjuvant. Test bleeds are taken approximately 7-10 days following each immunization and used to monitor the titer of the antiserum by ELISA.

[0304] To test serum, such as rabbit serum, for reactivity with 103P2D6 proteins, the full-length 103P2D6 cDNA can be cloned into an expression vector such as one that provides a 6His tag at the carboxyl-terminus (pcDNA 3.1 myc-his, Invitrogen). After transfection of the constructs into 293T cells, cell lysates can be probed with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.) and the anti-103P2D6 serum using Western blotting. Alternatively specificity of the antiserum is tested by Western blot and immunoprecipitation analyses using lysates of cells that express 103P2D6. Serum from rabbits immunized with GST or MBP fusion proteins is first semi-purified by removal of anti-GST or anti-MBP antibodies by passage over GST and MBP protein columns respectively. Sera from His-tagged protein and peptide immunized rabbits as well as depleted GST and MBP protein sera are purified by passage over an affinity column composed of the respective immunogen covalently coupled to Affigel matrix (BioRad).

Example 5 Production of Recombinant 103P2D6 in Bacterial and Mammalian Systems

[0305] Bacterial Constructs

[0306] pGEX Constructs

[0307] To express 103P2D6 in bacterial cells, portions of 103P2D6 were fused to the glutathione S-transferase (GST) gene by cloning into pGEX-6P-1 (Amersham Pharmacia Biotech, NJ). The constructs were made in order to generate recombinant 103P2D6 protein sequences with GST fused at the N-terminus and a six histidine epitope at the C-terminus. The six histidine epitope tag is generated by adding the histidine codons to the cloning primer at the 3′ end of the open reading frame (ORF). A PreScission™ recognition site permits cleavage of the GST tag from 103P2D6-related protein. The ampicillin resistance gene and pBR322 origin permits selection and maintenance of the plasmid in E. coli. For example, cDNA encoding the following fragments of 103P2D6 protein were cloned into pGEX-6P-1: amino acids 24 to 487; amino acids 39 to 176; amino acids 170 to 360; and amino acids 1 to 536, or any 8, 9, 10, 11, 12, 13, 14 or 15 contiguous amino acids from 103P2D6 or an analog thereof.

[0308] pMAL Constructs

[0309] To express 103P2D6in bacterial cells, all or part of the 103P2D6nucleic acid sequence are fused to the maltose-binding protein (MBP) gene by cloning into pMAL-c2X and pMAL-p2X (New England Biolabs, MA). The constructs are made to generate recombinant 103P2D6protein sequences with MBP-fused at the N-terminus and a six histidine epitope at the C-terminus. The six histidine epitope tag is generated by adding the histidine codons to the 3′ cloning primer. A Factor Xa recognition site permits cleavage of the GST tag from 103P2D6. The pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the cytoplasm or periplasm respectively. Periplasm expression enhances folding of proteins with disulfide bonds. For example, cDNA encoding the following fragments of 103P2D6 protein are cloned into pGEX-6P-1: amino acids 24 to 487; amino acids 39 to 176; amino acids 170 to 360; and amino acids 1 to 536, or any 8, 9, 10, 11, 12, 13, 14 or 15 contiguous amino acids from 103P2D6 or an analog thereof.

[0310] pCRII

[0311] To generate 103P2D6 sense and anti-sense riboprobes for RNA in situ investigations, a pCRII construct was generated using cDNA sequence encoding amino acids 44-181. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the production of 108P5H8 RNA riboprobes which will be used in RNA in situ hybridization experiments.

[0312] Mammalian Constructs

[0313] To express recombinant 103P2D6, the full or partial length 103P2D6cDNA can be cloned into any one of a variety of expression vectors known in the art. The constructs can be transfected into any one of a wide variety of mammalian cells such as 293T cells. Transfected 293T cell lysates can be probed with the anti-103P2D6polyclonal serum, described in Example 4 above, in a Western blot.

[0314] The 103P2D6genes can also be subcloned into the retroviral expression vector pSRαMSVtkneo and used to establish 103P2D6-expressing cell lines as follows: The 103P2D6 coding sequence (from translation initiation ATG and Kozak translation start consensus sequence to the termination codons) is amplified by PCR using ds cDNA template from 103P2D6 cDNA. The PCR product is subcloned into pSRαMSVtkneo vector and transformed into DH5α competent cells. Colonies are picked to screen for clones with unique internal restriction sites on the cDNA. The positive clone is confirmed by sequencing of the cDNA insert. The retroviral vectors can thereafter be used for infection and generation of various cell lines using, for example, NIH 3T3, TsuPr1, 293 or rat-1 cells.

[0315] Additional illustrative mammalian and bacterial systems are discussed below. pcDNA4/HisMax-TOPO Constructs To express 103P2D6 in mammalian cells, the 103P2D6 ORF is cloned into pcDNA4/HisMax-TOPO Version A (cat# K864-20, Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP163 translational enhancer. The recombinant protein has Xpress™ and six histidine epitopes fused to the N-terminus to aid in detection and purification of the recombinant protein. The pcDNA4/HisMax-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli.

[0316] pcDNA3.1/MycHis Constructs

[0317] To express 103P2D6 in mammalian cells, the ORF with consensus Kozak translation initiation site is cloned into pcDNA3.11MycHis_Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant protein has the myc epitope and six histidines fused to the C-terminus to aid in detection and purification of the recombinant protein. The pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability, along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene can be used, as it allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli.

[0318] pcDNA 3.1/V5His-TOPO Constructs

[0319] To express 103P2D6 in mammalian cells, the cDNA encoding the 103P2D6 ORF and Kozak consensus translation initiation sequence was cloned into pcDNA4NV5His-TOPO (cat# K4800-01, Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant protein has V₅™ and six histidine epitopes fused at the C-terminus to aid in detection and purification of the recombinant protein. The pcDNA4/V5His-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli.

[0320] Protein Expression

[0321] The mammalian expression vector pcDNA 3.1 VS-His encoding the 103P2D6 cDNA was used to transfect 293T human embryonic kidney cells to assess 103P2D6 protein expression. Western analysis of cell lysates using an anti-103P2D6 pAb reveals an immoreactive band of approximately 85 kD seen in 293T cells transiently transfected with the 103P2D6 cDNA but not in cells transfected with the control empty vector (FIG. 12A). Flow cytometric analysis of the same cells stained with the anti-103P2D6 pAb reveals a fluorescent shift of the 103P2D6 transfected cells compared to control cells indicating cell surface expression of the 103P2D6 protein (FIG. 12B). Western analysis of 103P2D6 mRNA positive HPAC and Bx PC-3 pancreatic cancer cells, CaCo-2 colon cancer cells, and LAPC9 prostate cancer cells, reveals expression of a 60 kD anti-103P2D6 immunoreactive band indicating significant endogenous expression of the 103P2D6 protein (Example 3, above; FIGS. 11A-B). The increased molecular weight of 103P2D6 in transfected 293T cells (85 kD) may be due to the presence of the VS-His amino acid tag present in the cDNA or post-translational processing or modification of 103P2D6 when overexpressed in 293T cells. 103P2D6 protein is expressed on the cell surface of Bx PC-3 cells as indicated by flow cytometric analysis (Example 3, above; FIG. 11C)

[0322] pcDNA3.1CT-GFP-TOPO Construct

[0323] To express 103P2D6 in mammalian cells and to allow detection of the recombinant protein using fluorescence, the ORF with consensus Kozak translation initiation site is cloned into pcDNA3.1CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant protein has the Green Fluorescent Protein (GFP) fused to the C-terminus facilitating non-invasive, in vivo detection and cell biology studies. The pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. An additional construct with a N-terminal GFP fusion is made in pcDNA3.1NT-GFP-TOPO spanning the entire length of the 103P2D6protein.

[0324] pAPtag Constructs

[0325] The cDNA encoding 103P2D6 amino acids 24-487 is cloned into pAPtag-5 (GenHunter Corp. Nashville, TN). This construct generates an alkaline phosphatase fusion at the C-terminus of the 103P2D6protein while fusing the IgGK signal sequence to N-terminus. The resulting recombinant 103P2D6 protein is optimized for secretion into the media of transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with the 103P2D6protein. Protein expression is driven from the CMV promoter and the recombinant protein also contains myc and six histidines fused to the C-terminus of alkaline phosphatase to aid in detection and purification of the recombinant protein. The Zeosin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene permits selection of the plasmid in E. coli.

[0326] ptag5 Constructs

[0327] The cDNA encoding for 103P2D6 amino acids 24-487, 24-174, 24-233, and 234-487, was cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates an immunoglobulin G1 Fc fusion at the C-terminus of the 103P2D6protein while fusing the IgGK signal sequence to the N-terminus. The resulting recombinant 103P2D6 protein is optimized for secretion into the media of transfected mammalian cells, and can be used to identify proteins such as ligands or receptors that interact with the 103P2D6protein. Protein expression is driven from the CMV promoter and the recombinant protein also contains myc and six histidines fused to the C-terminus to aid in detection and purification of the recombinant protein. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.

[0328] psecFc Constructs

[0329] The cDNA encoding for 103P2D6 amino acids 24-487, 24-233, and 234-487, was cloned into psecFc. The psecFc vector was assembled by cloning immunoglobulin G1 Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California). This construct generates an immunoglobulin G1 Fc fusion at the C-terminus of the 103P2D6protein, while fusing the IgGK signal sequence to N-terminus. The resulting recombinant 103P2D6 protein is optimized for secretion into the media of transfected mammalian cells, and can be used to identify proteins such as ligands or receptors that interact with the 103P2D6 protein. Protein expression is driven from the CMV promoter. The Zeocin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.

[0330] pSRαConstructs

[0331] To generate mammalian cell lines that express 103P2D6 constitutively, the ORF was cloned into pSRα constructs. Amphotropic and ecotropic retroviruses are generated by transfection of pSRα constructs into the 293T-10A1 packaging line or co-transfection of pSRα and a helper plasmid (φ˜) in the 293 cells, respectively. The retrovirus can be used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, 103P2D6, into the host cell-lines. Protein expression is driven from a long terminal repeat (LTR). The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permit selection and maintenance of the plasmid in E. coli.

[0332] An additional pSRα construct was made that fused the FLAG tag to the C-terminus to allow detection using anti-FLAG antibodies. The FLAG sequence 5′ gat tac aag gat gac gac gat aag 3′ (SEQ ID NO: XX) was added to cloning primer at the 3′ end of the ORF.

[0333] Additional pSRα constructs are made to produce both N-terminal and C-terminal GFP and myc/6 HIS fusion proteins of the full-length 103P2D6protein.

Example 6 Production of Recombinant 103P2D6in a Baculovirus System

[0334] To generate a recombinant 103P2D6 protein in a baculovirus expression system, cDNA sequence encoding the 103P2D6 protein is cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus Specifically, pBlueBac—103P2D6 is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supernatant and purified by plaque assay.

[0335] Recombinant 103P2D6protein is then generated by infection of HighFive insect cells (Invitrogen) with the purified baculovirus. Recombinant 103P2D6 protein can be detected using anti-103P2D6 antibody. 103P2D6 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 103P2D6.

Example 7 Chromosomal Mapping of the 103P2D6 Gene

[0336] The chromosomal localization of 103P2D6 was determined using the GeneBridge4 Human/Hamster radiation hybrid (RH) panel (Walter et al., 1994, Nat. Genetics 7:22) (Research Genetics, Huntsville Ala.).

[0337] The following PCR primers were used to localize 103P2D6:

[0338] 103P2D6.1 cftgggaggtcctagtgctaagtg

[0339] 103P2D6.2 caatgaagggactaacaacccatc

[0340] The resulting mapping vector for the 93 radiation hybrid panel DNAs was: 0100011101011010011011111100112001001000010011100111110010001011011001001101101101 10101000011. This vector and the mapping program at http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.p1 placed 103P2D6 on chromosome 4p12-p14 (D4S3197-D4S1577).

Example 8 Identification of Potential Signal Transduction Pathways

[0341] Based on its surface localization, 103P2D6 can regulate key signaling pathways. Several pathways known to play a role in cancer biology can be regulated by 103P2D6, including phospholipid pathways such as P13K, AKT, etc, as well as mitogenic/survival cascades such as ERK, p38, etc (Cell Growth Differ. 2000,11:279; J. Biol. Chem. 1999, 274:801; Oncogene. 2000, 19:3003.). The role that 103P2D6 plays in the regulation of these pathways can be investigated using, Western blotting techniques and reporter assays. Cells lacking 103P2D6 and cells expressing 103P2D6 are either left untreated or stimulated with serum, cytokines, androgen or antibodies. Cell lysates are analyzed using anti-phosphos-specific antibodies (Cell Signaling, Santa Cruz Biotechnology) in order to detect phosphorylation and regulation of ERK, p38, AKT, P13K, PLC and other signaling molecules. When 103P2D6 plays a role in the regulation of signaling pathways, 103P2D6 is used as a target for diagnostic, preventative and therapeutic purposes.

[0342] To determine whether 103P2D6 directly or indirectly activates known signal transduction pathways in cells, luciferase (luc) based transcriptional reporter assays are carried out in cells that express 103P2D6. These transcriptional reporters contain consensus binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways. Some of the reporters and examples of these associated transcription factors, signal transduction pathways, and activation stimuli are listed below.

[0343] 1. NFkB-luc, NFkB/Rel; Ilk-kinase/SAPK: growth/apoptosis/stress

[0344] 2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK: growth/differentiation

[0345] 3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC: growth/apoptosis/stress

[0346] 4. ARE-luc, androgen receptor; steroids/MAPK: growth/differentiation/apoptosis

[0347] 5. p53-luc, p53; SAPK: growth/differentiation/apoptosis

[0348] 6. CRE-luc, CREB/ATF2: PKA/p38; growth/apoptosis/stress

[0349] When 103P2D6 plays a role in the regulation of growth, apoptosis, stress, or differentiation, 103P2D6 is used as a target for diagnostic, preventative and therapeutic purposes.

[0350] 103P2D6-mediated effects are assayed in cells showing 103P2D6 mRNA expression. For example, Luciferase reporter plasmids can be introduced by lipid-mediated transfection (TFX-50, Promega). Luciferase activity, an indicator of relative transcriptional activity, is measured by incubation of cell extracts with luciferin substrate and luminescence of the reaction is monitored in a luminometer. This assay allows one to determine the effect of signaling pathways activation. When 103P2D6 plays a role in activation signaling pathways, 103P2D6 is used as a target for diagnostic, preventative and therapeutic purposes.

Example 9 Generation of 103P2D6 Monoclonal Antibodies (mAbs)

[0351] To generate mAbs to 103P2D6, mice are immunized intraperitoneally with 10-50 μg of protein immunogen mixed in complete Freund's adjuvant. Protein immunogens include peptides, recombinant 103P2D6 proteins, and, mammalian expressed human IgG FC fusion proteins. Mice are then subsequently immunized every 2-4 weeks with 10-50 μg of antigen mixed in Freund's incomplete adjuvant. Alternatively, Ribi adjuvant is used for initial immunizations. In addition, a DNA-based immunization protocol is used in which a mammalian expression vector used to immunize mice by direct injection of the plasmid DNA. For example, a pcDNA 3.1 encoding 103P2D6 cDNA alone or as an IgG FC fusion is used. This protocol is used alone or in combination with protein immunogens. Test bleeds are taken 7-10 days following immunization to monitor titer and specificity of the immune response. Once appropriate reactivity and specificity is obtained as determined by ELISA, Western blotting, and immunoprecipitation analyses, fusion and hybridoma generation is then carried with established procedures well known in the art (Harlow and Lane, 1988).

[0352] In an illustrative method for generating 103P2D6 monoclonal antibodies, a glutathione-S-transferase (GST) fusion protein encompassing a 103P2D6 protein is synthesized and used as immunogen. Balb C mice are initially immunized intraperitoneally with 200 μg of the GST-103P2D6 fusion protein mixed in complete Freund's adjuvant. Mice are subsequently immunized every two weeks with 75 μg of GST-103P2D6 protein mixed in Freund's incomplete adjuvant for a total of three immunizations. Reactivity of serum from immunized mice to full-length 103P2D6 protein is monitored by ELISA using a partially purified preparation of HIS-tagged 103P2D6 protein expressed from 293T cells (Example 5). Mice showing the strongest reactivity are rested for three weeks and given a final injection of fusion protein in PBS and then sacrificed four days later. The spleens of the sacrificed mice are then harvested and fused to SPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988). Supernatants from growth wells following HAT selection are screened by ELISA and Western blot to identify 103P2D6 specific antibody-producing clones.

[0353] The binding affinity of a 103P2D6 monoclonal antibody is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and can be used to help define which 103P2D6 monoclonal antibodies are preferred for diagnostic or therapeutic use. The BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity. The BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.

Example 10 Involvement of 103P2D6 in Cancer Growth and Progression

[0354] The finding that 103P2D6 is differentially expressed in cancer cells suggests that 103P2D6 participates in the process of tumor formation and/or progression. This is supported by the presence of other envelope-like proteins in prostate, colon and endothelial tumor lines but not normal cells (Pathobiology. 1997, 65:123; Cancer Lett. 1998, 124:213). For example, HERV-K, another envelope protein, is expressed in teratocarcinoma and tumor cell lines (3 Gen Virol. 1996; 77: 2983), and HERV-R is expressed in gonadoblastomas (Virchows Arch. 1999; 434:11).

[0355] 103P2D6 contributes to the growth of prostate and other cancer cells (See Table I) by several mechanisms. The 103P2D6 protein can function as a cell surface receptor or as a transporter and contribute to tumor growth by regulating tumor cell proliferation or responding to tumor cells, endothelial cells or stroma. Alternatively, 103P2D6 contributes to tumor growth by mediating cellular adhesion, transformation or downstream gene expression. The functions of 103P2D6 can be evaluated, e.g., by using engineered cell lines that express 103P2D6. For example, primary cells such as PrEC, cancer cell lines and NIH 3T3 cells engineered to stably express 103P2D6 are evaluated for cell growth potential. When 103P2D6 participates in neoplastic cell growth, 103P2D6 is used as a target for diagnostic, preventative and therapeutic purposes.

[0356] Moreover, the role 103P2D6 plays in transformation is evaluated. Primary cells, such as PrEC, and NIH3T3 cells engineered to express 103P2D6 are compared to 103P2D6-negative cells, to evaluate their ability to form colonies in soft agar (Song Z. et al. Cancer Res. 2000; 60:6730), where colony formation indicates the presence of transformed cells. When 103P2D6 mediates transformation, 103P2D6 is used as a target for diagnostic, preventative and therapeutic purposes.

[0357] The expression of 103P2D6 in the various cancers listed in Table I indicates that this gene has a functional role in tumor progression. 103P2D6 function can be assessed in mammalian cells using in vitro approaches. Mammalian cells infected with the retroviral vector pSRαtkneo or pSRα-103P2D6 are compared (Muller et al., 1991, MCB 11:1785) using a variety of assays, including cell proliferation in tissue culture and in vitro invasion using a membrane invasion culture system (Welch et al., Int. J. Cancer 43: 449-457). Cell lines expressing 103P2D6 are assayed for invasive and migratory properties by measuring passage of cells through a matrigel coated Transwell™ system (Becton Dickinson) (Cancer Res. 1999; 59:6010). Passage of cells through the membrane to the opposite side is monitored using a fluorescent assay (Becton Dickinson Technical Bulletin #428). For example, cells lacking 103P2D6 and cells expressing 103P2D6 are loaded with the fluorescent dye, calcein, and plated in the top well of the Transwell insert. Invasion is determined by fluorescence of cells in the lower chamber relative to the fluorescence of the entire cell population. When 103P2D6 is involved with cell invasion, 103P2D6 is used as a target for diagnostic, preventative and therapeutic purposes.

[0358] Envelope proteins have been shown to exhibit chemotactic abilities (Lin C L, Sewell A K, Gao fGF, Whelan K T, Phillips R E, Austyn J M. J Exp Med. 2000, 192:587). In view of its similarity to envelope proteins, and to determine whether 103P2D6-expressing cells have such chemoattractant properties, indicator cells are monitored for passage through the Transwell system toward a gradient of 103P2D6-conditioned media compared to control media. This assay can also be used to qualify and quantify specific neutralization of 103P2D6 effects. For example, the neutralization can be effected by candidate cancer therapeutic compositions. When 103P2D6 mediates tissue invasion, such as by chemotaction, 103P2D6 is used as a target for diagnostic, preventative and therapeutic purposes.

[0359] The function of 103P2D6 can be evaluated using anti-sense RNA technology coupled to the various functional assays described herein, e.g. growth, invasion and migration. Anti-sense RNA oligonucleotides can be introduced into 103P2D6 expressing cells, thereby preventing the expression of 103P2D6. Control and anti-sense containing cells can be analyzed for proliferation, invasion, migration, apoptotic and transcriptional potential. The local as well as systemic effect of the loss of 103P2D6 expression can be evaluated.

Example 11 Protein Association and Cell Adhesion

[0360] Envelope proteins have been shown to mediate cell adhesion and syncytium formation (J. Immunol. 1996, 156:1307; AIDS. 1991, 5:1425). Based on its similarity with envelope proteins, 103P2D6 can mediate protein-protein association resulting in multimeric complexes. The association of proteins into large complexes is critical in several biological processes, including signal transduction, cell communication, ubiquitination, transcriptional regulation, etc.

[0361] Alternatively, 103P2D6 can participate in regulating cell adhesion and communication. To determine the degree to which expression of 103P2D6 regulates cell adhesion, cells lacking 103P2D6 are compared to cells expressing 103P2D6, using techniques known in the art (see, e.g., Haier et al, Br. J. Cancer. 1999, 80:1867; Lehr and Pienta, J. Natl. Cancer Inst. 1998, 90:118). Briefly, in one embodiment, cells labeled with a fluorescent indicator, such as calcein, are incubated on tissue culture wells coated with media alone or with matrix proteins. Adherent cells are detected by fluorimetric analysis. Confirmation of the role 103P2D6 plays in adhesion can be obtained using anti-103P2D6 antibodies. Since cell adhesion plays a critical role in tumor growth, progression, and, colonization, the inhibition of 103P2D6-mediated interactions serves as a diagnostic, preventative and therapeutic modality.

Example 12 In vivo Assay for 103P2D6 Tumor Growth Promotion

[0362] The effect of the 103P2D6 protein on tumor cell growth can be evaluated in vivo by gene overexpression in tumor-bearing mice. For example, SCID mice can be injected SQ on each flank with 1×10⁶ of either PC3, TSUPR1, or DU145 cells containing tkneo empty vector or 103P2D6. At least two strategies may be used: (1) Constitutive 103P2D6 expression under regulation of a promoter such as a constitutive promoter obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UTK 2,211,504 published Jul. 5, 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, provided such promoters are compatible with the host cell systems. (2) Regulated expression under control of an inducible vector system, such as ecdysone, tet, etc., can be used provided such promoters are compatible with the host cell systems. Tumor volume is then monitored at the appearance of palpable tumors and is followed over time to determine if 103P2D6-expressing cells grow at a faster rate and whether tumors produced by 103P2D6-expressing cells demonstrate characteristics of altered aggressiveness (e.g. enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs). Additionally, mice can be implanted with 1×10⁵ of the same cells orthotopically to determine if 103P2D6 has an effect on local growth in the prostate or on the ability of the cells to metastasize, specifically to lungs, lymph nodes, and bone marrow. Also see saffron et al, “Anti-PSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival of mice bearing human prostate cancer xenografts” PNAS (in press, 2001).

[0363] The assay is also useful to determine the 103P2D6 inhibitory effect of candidate therapeutic compositions, such as for example, 103P2D6 intrabodies, 103P2D6 antisense molecules and ribozymes.

Example 13 Western Analysis of 103P2D6 Expression in Subcellular Fractions

[0364] The cellular location of 103P2D6 can be assessed using subcellular fractionation techniques widely used in cellular biology (Storrie B, et al. Methods Enzymol. 1990; 182:203-25). Prostate or other cell lines can be separated into nuclear, cytosolic and membrane fractions. The expression of 103P2D6 in the different fractions can be tested using Western blotting techniques.

[0365] Alternatively, to determine the subcellular localization of 103P2D6, 293T cells can be transfected with an expression vector encoding HIS-tagged 103P2D6 (PCDNA 3.1 MYC/HIS, Invitrogen). The transfected cells can be harvested and subjected to a differential subcellular fractionation protocol as previously described (Pemberton, P. A. et al, 1997, J of Histochemistry and Cytochemistry, 45:1697-1706.) This protocol separates the cell into fractions enriched for nuclei, heavy membranes (lysosomes, peroxisomes, and mitochondria), light membranes (plasma membrane and endoplasmic reticulum), and soluble proteins.

Example 14 Localization of 103P2D6

[0366] Based on its structure, 103P2D6 is understood to be associated with the cell surface. Surface staining experiments confirm that 103P2D6 cDNA is expressed at the cell surface (see, e.g., FIGS. 11-12). When located at the cell membrane, the potential functions of 103P2D6 include (1) a surface receptor that transmits signals to the cell nucleus, or (2) a transporter that moves ions and proteins from in and out of the cell, or (3) a mediator of cell adhesion and cell-cell interaction. The cellular location of 103P2D6 can be assessed using subcellular fractionation techniques widely used in cellular biology (see, e.g., Storrie B, et al. Methods Enzymol. 1990; 182:203-25). Prostate, colon, bladder, kidney or pancreas tumor cell lines are separated into nuclear, cytosolic, heavy membranes (lysosomes, peroxisomes, and mitochondria) and light membranes (plasma membrane and endoplasmic reticulum) fractions. The expression of 103P2D6 is followed in each fraction by Western blotting. When 103P2D6 participates in cell adhesion or cell-cell communication, 103P2D6 is used as a target for diagnostic, preventative and therapeutic purposes.

Example 15 Protein-Protein Interactions Mediated by 103P2D6

[0367] The determination of the specific proteins with which 103P2D6 associates, including cytoskeleton and integrins, can be made, e.g., using co-precipitation and Western blotting techniques (see, e.g., Hamilton B J, et al. Biochem Biophys. Res. Commun. 1999, 261:646). Immunoprecipitates from cells expressing 103P2D6 and cells lacking 103P2D6 are compared for specific protein-protein associations. 103P2D6 also associates with effector molecules, such as C2-domain containing proteins. Studies comparing 103P2D6 positive and 103P2D6 negative cells as well as studies comparing unstimulated/resting cells and cells treated with epithelial cell activators, such as cytokines, androgen and antibodies reveal unique interactions. In addition, protein-protein interactions can be studied using two yeast hybrid methodology (see, e.g., Curr Opin Chem Biol. 1999, 3:64). A vector carrying a library of proteins fused to the activation domain of a transcription factor is introduced into yeast expressing a 103P2D6-DNA-binding domain fusion protein and a reporter construct. Protein-protein interaction is detected by calorimetric reporter activity. Specific association with effector molecules and transcription factors directs one of skill to the mode of action of 103P2D6, and thus identifies therapeutic, preventative and/or diagnostic targets for cancer.

Example 16 Regulation of Transcription by 103P2D6

[0368] The 103P2D6 protein contains a leucine zipper at its amino-terminus. Leucine zippers play a role in protein dimerization and determine sequence-specific DNA binding (Luscher B, Larsson L G. Oncogene. 1999; 18:2955), and are therefore transcriptional regulators. Analogously, 103P2D6 can regulate tumor progression by regulating gene expression. The role that 103P2D6 plays in regulating gene expression can be evaluated, e.g., by studying gene expression in cells expressing or lacking 103P2D6. For example, RNA from parental and 103P2D6-expressing NIH3T3 and PC3 cells is extracted and hybridized to commercially available gene arrays (Clontech). Resting cells as well as cells treated with serum, cytokines, androgen or antibodies are compared. Differentially expressed genes are identified and mapped to biological pathways. When 103P2D6 regulates transcription, 103P2D6 is used as a target for diagnostic, preventative and therapeutic purposes.

Example 17 103P2D6 Monoclonal Antibody Mediated Inhibition of Prostate Tumors in vivo

[0369] The significant expression of 103P2D6, in cancer tissues, together with its restrictive expression in normal tissues makes 103P2D6 a target for antibody therapy. Similarly, 103P2D6 is a target for T cell-based immunotherapy. Thus, the therapeutic efficacy of anti-103P2D6 mAbs in human prostate cancer xenograft mouse models is evaluated by using the androgen-dependent LAPC-9 xenograft (Craft, N., et al., Cancer Res, 1999. 59(19): p. 5030-6) and the androgen independent recombinant cell line PC3-103P2D6 (see, e.g., Kaighn, M. E., et al., Invest Urol, 1979. 17(1): p. 16-23).

[0370] Antibody efficacy on tumor growth and metastasis formation is studied, e.g., in a mouse orthotopic prostate cancer xenograft model. These studies demonstrate that anti-103P2D6 MAbs inhibit formation of both the androgen-dependent LAPC-9 and androgen-independent PC3-103P2D6 tumor xenografts. Anti-103P2D6 mAbs also retard the growth of established orthotopic tumors significantly and prolonged survival of tumor-bearing mice. These results indicate the utility of anti-103P2D6 mAbs in the treatment of local and advanced stages of prostate cancer.

[0371] Administration of the anti-103P2D6 mAbs led to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate that 103P2D6 as an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-103P2D6 mAbs for the treatment of local and metastatic prostate cancer. This example demonstrates that unconjugated 103P2D6 monoclonal antibodies are effective to inhibit the growth of human prostate tumor xenografts grown in SCID mice; accordingly a combination of such efficacious monoclonal antibodies is also effective.

[0372] Tumor Inhibition using Multiple Unconjugated 103P2D6 mAbs

[0373] Materials and Methods

[0374] 103P2D6 Monoclonal Antibodies:

[0375] Monoclonal antibodies are raised against 103P2D6 as described in Example 9.

[0376] Antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their capacity to bind 103P2D6. Epitope mapping data for the anti-103P2D6 mAbs, as determined by ELISA and Western analysis, recognize epitopes on the 103P2D6 protein. Immunohistochemical analysis of prostate cancer tissues and cells with these antibodies is performed.

[0377] Antibody Formulation:

[0378] The monoclonal antibodies are purified from hybridoma tissue culture supernatants by Protein-G Sepharose chromatography, dialyzed against PBS, filter sterilized, and stored at −20° C. Protein determinations are performed by a Bradford assay (Bio-Rad, Hercules, Calif.). A therapeutic monoclonal antibody or a cocktail comprising a mixture of individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or orthotopic injections of LAPC-9 prostate tumor xenografts.

[0379] Prostate Cancer Xenografts and Cell Lines.

[0380] The LAPC-9 xenograft, which expresses a wild-type androgen receptor and produces prostate-specific antigen (PSA), is passaged in 6- to 8-week-old male ICR-severe combined imiunodeficient (SCID) mice (Taconic Farms) by s.c. trocar implant (Craft, N., et al., supra). Single-cell suspensions of LAPC-9 tumor cells are prepared as described in Craft, et al. The prostate carcinoma cell line PC3 (American Type Culture Collection) is maintained in DMEM supplemented with L-glutamine and 10% (vol/vol) FBS.

[0381] A PC3-103P2D6 cell population is generated by retroviral gene transfer as described in Hubert, R. S., et al., Proc Natl Acad Sci USA, 1999. 96(25): p. 14523-8. Anti-103P2D6 staining is detected by using an FITC-conjugated goat anti-mouse antibody (Southern Biotechnology Associates) followed by analysis on a Coulter Epics-XL flow cytometer.

[0382] Xenograft Mouse Models.

[0383] Subcutaneous (s.c.) tumors are generated by injection of 1×10⁶ LAPC-9, PC3, or PC3-103P2D6 cells mixed at a 1:1 dilution with Matrigel (Collaborative Research) in the right flank of male SCID mice. To test antibody efficacy on tumor formation, i.p. antibody injections are started on the same day as tumor-cell injections. As a control, mice are injected with either purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody that recognizes an irrelevant antigen not expressed in human cells. In preliminary studies, no difference is found between mouse IgG or PBS on tumor growth. Tumor sizes are determined by vernier caliper measurements, and the tumor volume is calculated as length×width×height. Mice with s.c. tumors greater than 1.5 cm in diameter are sacrificed. PSA levels are determined by using a PSA ELISA kit (Anogen, Mississauga, Ontario). Circulating levels of anti-103P2D6 mAbs are determined by a capture ELISA kit (Bethyl Laboratories, Montgomery, Tex.).

[0384] Orthotopic injections are performed under anesthesia by using ketamine/xylazine. An incision is made through the abdominal muscles to expose the bladder and seminal vesicles, which then are delivered through the incision to expose the dorsal prostate. LAPC-9 cells (5×10⁵) mixed with Matrigel are injected into each dorsal lobe in a 10-μl volume. To monitor tumor growth, mice are bled on a weekly basis for determination of PSA levels. Based on the PSA levels, the mice are segregated into groups for the appropriate treatments. To test the effect of anti-103P2D6 mAbs on established orthotopic tumors, i.p. antibody injections are started when PSA levels reach 2-80 ng/ml.

[0385] Anti-103P2D6 mAbs Inhibit Formation of 103P2D6-Expressing Prostate-Cancer Tumors

[0386] We next test the effect of anti-103P2D6 mAbs on tumor formation by using the LAPC-9 orthotopic model. As compared with the s.c. tumor model, the orthotopic model, which requires injection of tumor cells directly in the mouse prostate, results in a local tumor growth, development of metastasis in distal sites, deterioration of mouse health, and subsequent death (Saffran, D., et al., PNAS 10:1073-1078 or www.pnas.org/cgi/doi/10.1073/pnas.051624698; Fu, X., et al., Int J Cancer, 1992. 52(6): p. 987-90; Kubota, T., J Cell Biochem, 1994. 56(1): p. 4-8). The features make the orthotopic model more representative of human disease progression and allowed us to follow the therapeutic effect of mAbs on clinically relevant end points.

[0387] Accordingly, LAPC-9 tumor cells are injected into the mouse prostate, and 2 days later, the mice are segregated into two groups and treated with either 200,g of 1G8 mAb or PBS three times per week for two weeks. Mice are monitored weekly for circulating PSA levels as an indicator of tumor growth.

[0388] A major advantage of the orthotopic prostate-cancer model is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studies by IHC analysis on lung sections using an antibody against a prostate-specific cell-surface protein STEAP expressed at high levels in LAPC-9 xenografts (Hubert, R. S., et al., Proc Natl Acad Sci USA, 1999. 96(25): p. 14523-8).

[0389] Mice bearing established orthotopic LAPC-9 tumors are administered 11 injections of either anti-103P2D6 mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden (PSA levels greater than 300 ng/ml), to ensure a high frequency of metastasis formation in mouse lungs. Mice then are killed and their prostate and lungs are analyzed for the presence of LAPC-9 cells by anti-STEAP IHC analysis.

[0390] These studies demonstrate a broad anti-tumor efficacy of anti-103P2D6 antibodies on initiation and progression of prostate cancer in xenograft mouse models. Anti-103P2D6 antibodies inhibit tumor formation of both androgen-dependent and androgen-independent tumors as well as retarding the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-103P2D6 mAbs demonstrate a dramatic inhibitory effect on the spread of local prostate tumor to distal sites, even in the presence of a large tumor burden. Thus, anti-103P2D6 mAbs are efficacious on major clinically relevant end points/PSA levels (tumor growth), prolongation of survival, and health.

[0391] Throughout this application, various publications are referenced (within parentheses for example). The disclosures of these publications are hereby incorporated by reference herein in their entireties.

[0392] The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention. TABLE I Cancer Tissues That Express 103P2D6 prostate colon stomach lung pancreas breast bladder bone kidney testis cervix ovary uterus

[0393] TABLE II AMINO ACID ABBREVIATIONS SINGLE LETTER THREE LETTER FULL NAME F Phe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cys cysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamine R Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asn asparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid E Glu glutamic acid G Gly glycine

[0394] TABLE III AMINO ACID SUBSTITUTION MATRIX Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix (block substi- tution matrix). The higher the value, the more likely a substitution is found in related, natural proteins. A C D E F G H I K L M N P Q R S T V W Y 4 0 −2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 A 9 −3 −4 −2 −3 −3 −1 −3 −1 −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 C 6 2 −3 −1 −1 −3 −1 −4 −3 1 −1 0 −2 0 −1 −3 −4 −3 D 5 −3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3 −2 E 6 −3 −1 0 −3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 F 6 −2 −4 −2 −4 −3 0 −2 −2 −2 0 −2 −3 −2 −3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 H 4 −3 2 1 −3 −3 −3 −3 −2 −1 3 −3 −1 I 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 K 4 2 −3 −3 −2 −2 −2 −1 1 −2 −1 L 5 −2 −2 0 −1 −1 −1 1 −1 −1 M 6 −2 0 0 1 0 −3 −4 −2 N 7 −1 −2 −1 −1 −2 −4 −3 P 5 1 0 −1 −2 −2 −1 Q 5 −1 −1 −3 −3 −2 R 4 1 −2 −3 −2 S 5 0 −2 −2 T 4 −3 −1 V 11 2 W 7 Y

[0395] TABLE IV A HLA CLASS I SUPERMOTIFS SUPERMOTIF POSITION 2 C-TERMINUS A2 L, I, V, M, A, T, Q L, . I, V, M, A, T A3 A, V, I, L, M, S, T R, K B7 P A, L, I, M, V, F, W, Y B44 D, E F, W, Y, L, I, M, V, A A1 T, S, L, I, V, M F, W, Y A24 F, W, Y, L, V, I, M, T F, I, Y, W, L, M B27 R, H, K A, L, I, V, M, Y, F, W B58 A, S, T F, W, Y, L, I, V B62 L, V, M, P, I, Q F, W, Y, M, I, V

[0396] TABLE IV B HLA CLASS II SUPERMOTIF 1 6 9 W, F, Y, V, .I, L A, V, I, L, P, C, S, T A, V, I, L, C, S, T, M, Y

[0397] TABLE V Scoring Results 103P2D6 HLA peptides A1 9MERS Score (Estimate of Half Time of Disassociation of Start Subsequence a Molecule Containing Rank Position Residue Listing This Subsequence) 1 52 NAEQPELVF 45.000 2 253 LTEAHGKWR 22.500 3 105 SMEAQGLSF 22.500 4 356 DTDNPPLYC 6.250 5 153 STDGTFMPS 6.250 6 161 SIDVTNESR 5.000 7 321 YLVPSLTRY 5.000 8 374 ALFPSLGTY 5.000 9 425 VLDIPTTQR 5.000 10 473 LLDWQGIFA 2.500 11 500 CLFIFVLIY 2.500 12 208 TQDYKWVDR 1.500 13 538 VSETSCQVS 1.350 14 450 YSEEIKSNI 1.350 15 203 IGLPNTQDY 1.250 16 69 WTYSGQWMY 1.250 17 506 LIYVRVFRK 1.000 18 434 QTACGTVGK 1.000 19 378 SLGTYDLEK 1.000 20 405 TLEAHQSKV 0.900 21 461 LHEASENLK 0.900 22 118 LLEGNFSLC 0.900 23 126 CVENKNGSG 0.900 24 84 QAEVQNHST 0.900 25 520 NSQPLNLAL 0.750 26 63 ASASTWWTY 0.750 27 222 WSGNDTCLY 0.750 28 233 QNQTKGLLY 0.625 29 21 LVQPQHLLA 0.500 30 404 QTLEAHQSK 0.500 31 136 FLGNIPKQY 0.500 32 496 IVLFCLFIF 0.500 33 291 SVNNSGLFF 0.500 34 383 DLEKAILNI 0.450 35 394 AMEQEFSAT 0.450 36 55 QPELVFVPA 0.450 37 299 FLCGNGVYK 0.400 38 313 WSGRCGLGY 0.375 39 442 KQCCLYINY 0.375 40 414 SSLASASRK 0.300 41 413 VSSLASASR 0.300 42 71 YSGQWMYER 0.300 43 134 GPFLGNIPK 0.250 44 354 QGDTDNPPL 0.250 45 174 DTSVCLGTR 0.250 46 224 GNDTCLYSC 0.250 47 540 ETSCQVSNR 0.250 48 150 WFDSTDGTF 0.250 49 502 FIFVLIYVR 0.200 50 505 VLIYVRVFR 0.200

[0398] TABLE VI Scoring Results 103P2D6 HLA peptides A1 10- MERS Score (Estimate of Half Time of Disassociation of Start Subsequence a Molecule Containing Rank Position Residue Listing This Subsequence) 1 473 LLDWQGIFAK 50.000 2 394 AMEQEFSATK 18.000 3 354 QGDTDNPPLY 12.500 4 262 CADASITNDK 10.000 5 483 VGDWFRSWGY 6.250 6 118 LLEGNFSLCV 4.500 7 464 ASENLKNVPL 2.700 8 538 VSETSCQVSN 2.700 9 153 STDGTFMPSI 2.500 10 356 DTDNPPLYCN 2.500 11 337 ITNLRSFIHK 2.500 12 499 FCLFIFVLIY 2.500 13 451 SEEIKSNIQR 2.250 14 253 LTEAHGKWRC 2.250 15 46 LCEHLDNAEQ 1.800 16 52 NAEQPELVFV 1.800 17 450 YSEEIKSNIQ 1.350 18 438 GTVGKQCCLY 1.250 19 505 VLIYVRVFRK 1.000 20 49 HLDNAEQPEL 1.000 21 84 QAEVQNHSTS 0.900 22 405 TLEAHQSKVS 0.900 23 104 ASMEAQGLSF 0.750 24 290 LSVNNSGLFF 0.750 25 203 IGLPNTQDYK 0.500 26 161 SIDVTNESRN 0.500 27 202 LIGLPNTQDY 0.500 28 425 VLDIPTTQRQ 0.500 29 358 DNPPLYCNPK 0.500 30 373 RALFPSLGTY 0.500 31 207 NTQDYKWVDR 0.500 32 495 LIVLFCLFIF 0.500 33 86 EVQNHSTSSY 0.500 34 198 SAVPLIGLPN 0.500 35 55 QPELVFVPAS 0.450 36 105 SMEAQGLSFA 0.450 37 542 SCQVSNRAMK 0.400 38 232 CQNQTKGLLY 0.375 39 413 VSSLASASRK 0.300 40 520 NSQPLNLALS 0.300 41 213 WVDRNSGLTW 0.250 42 307 KGFPPKWSGR 0.250 43 133 SGPFLGNIPK 0.250 44 461 LHEASENLKN 0.225 45 412 KVSSLASASR 0.200 46 504 FVLIYVRVFR 0.200 47 460 RLHEASENLK 0.200 48 228 CLYSCQNQTK 0.200 49 527 ALSPQQSAQL 0.200 50 377 PSLGTYDLEK 0.150

[0399] TABLE VII Scoring Results 103P2D6 HLA peptides A2 9MERS Score (Estimate of Half Time of Disassociation of Start Subsequence a Molecule Containing Rank Position Residue Listing This Subsequence) 1 497 VLFCLFIFV 4571.688 2 117 RLLEGNFSL 1879.592 3 493 VLLIVLFCL 1792.489 4 45 WLCEHLDNA 105.596 5 282 WLTGSNLTL 98.267 6 492 YVLLIVLFC 93.592 7 13 TLTAFLTIL 76.550 8 10 LQLTLTAFL 74.930 9 75 WMYERVWYP 61.634 10 495 LIVLFCLFI 50.968 11 79 RVWYPQAEV 50.512 12 318 GLGYLVPSL 49.134 13 329 YLTLNASQI 47.991 14 241 YQLFRNLFC 47.151 15 339 NLRSFIHKV 46.199 16 239 LLYQLFRNL 44.160 17 11 QLTLTAFLT 43.222 18 460 RLHEASENL 42.917 19 537 LVSETSCQV 42.418 20 20 ILVQPQHLL 36.316 21 228 CLYSCQNQT 23.846 22 110 GLSFAQVRL 21.362 23 518 SLNSQPLNL 21.362 24 289 TLSVNNSGL 21.362 25 393 KAMEQEFSA 21.249 26 473 LLDWQGIFA 18.580 27 535 QLLVSETSC 18.382 28 8 ALLQLTLTA 18.382 29 292 VNNSGLFFL 17.393 30 53 AEQPELVFV 17.108 31 27 LLAPVFRTL 13.800 32 108 AQGLSFAQV 13.398 33 26 HLLAPVFRT 12.506 34 19 TILVQPQHL 10.868 35 119 LEGNFSLCV 8.737 36 336 QITNLRSFI 7.890 37 219 GLTWSGNDT 7.452 38 552 GLTTHQYDT 7.452 39 246 NLFCSYGLT 5.377 40 405 TLEAHQSKV 4.451 41 242 QLFRNLFCS 3.678 42 238 GLLYQLFRN 3.678 43 156 GTFMPSIDV 3.574 44 322 LVPSLTRYL 3.495 45 38 LTNQSNCWL 2.774 46 36 SILTNQSNC 2.527 47 387 AILNISKAM 2.527 48 499 FCLFIFVLI 2.202 49 106 MEAQGLSFA 2.077 50 490 WGYVLLIVL 1.936

[0400] TABLE VIII Scoring Results 103P2D6 HLA peptides A2 10-MERS Score (Estimate of Half Time of Disassociation of Start Subsequence a Molecule Containing Rank Position Residue Listing This Subsequence) 1 500 CLFIFVLIYV 3255.381 2 497 VLFCLFIFVL 2792.698 3 492 YVLLIVLFCL 424.398 4 496 IVLFCLFIFV 400.023 5 494 LLIVLFCLFI 370.677 6 296 GLFFLCGNGV 257.342 7 502 FIFVLIYVRV 244.154 8 37 ILTNQSNCWL 199.738 9 9 LLQLTLTAFL 199.738 10 117 RLLEGNFSLC 143.193 11 291 SVNNSGLFFL 137.144 12 136 FLGNIPKQYC 125.690 13 536 LLVSETSCQV 118.238 14 321 YLVPSLTRYL 108.094 15 13 TLTAFLTILV 69.552 16 148 ILWFDSTDGT 51.522 17 331 TLNASQITNL 49.134 18 3 SLSNCALLQL 49.134 19 238 GLLYQLFRNL 30.036 20 118 LLEGNFSLCV 28.756 21 22 VQPQHLLAPV 27.573 22 10 LQLTLTAFLT 27.564 23 527 ALSPQQSAQL 21.362 24 11 QLTLTAFLTI 19.822 25 445 CLYTNYSEEI 16.359 26 338 TNLRSFIHKV 14.682 27 404 QTLEAHQSKV 14.654 28 329 YLTLNASQIT 14.054 29 518 SLNSQPLNLA 11.426 30 19 TILVQPQHLL 10.868 31 393 KAMEQEFSAT 10.441 32 110 GLSFAQVRLL 9.827 33 472 PLLDWQGIFA 9.119 34 353 TQGDTDNPPL 8.880 35 20 ILVQPQHLLA 8.446 36 318 GLGYLVPSLT 7.452 37 12 LTLTAFLTIL 6.687 38 205 LPNTQDYKWV 6.368 39 335 SQITNLRSFI 5.818 40 378 SLGTYDLEKA 5.599 41 439 TVGKQCCLYI 5.021 42 234 NQTKGLLYQL 4.982 43 325 SLTRYLTLNA 4.968 44 525 NLALSPQQSA 4.968 45 544 QVSNRAMKGL 4.299 46 220 LTWSGNDTCL 4.186 47 58 LVFVPASAST 4.101 48 488 RSWGYVLLIV 3.554 49 61 VPASASTWWT 3.411 50 343 FIHKVTPHRC 3.142

[0401] TABLE IX Scoring Results 103P2D6 HLA PEPTIDES A3 9-MERS Score (Estimate of Half Time of Disassociation of Start Subsequence a Molecule Containing Rank Position Residue Listing This Subsequence) 1 500 CLFIFVLIY 360.000 2 378 SLGTYDLEK 120.000 3 304 GVYKGFPPK 90.000 4 506 LIYVRVFRK 90.000 5 204 GLPNTQDYK 60.000 6 299 FLCGNGVYK 30.000 7 321 YLVPSLTRY 13.500 8 374 ALFPSLGTY 13.500 9 505 VLIYVRVFR 9.000 10 494 LLIVLFCLF 9.000 11 502 FIFVLIYVR 9.000 12 493 VLLIVLFCL 6.075 13 425 VLDIPTTQR 6.000 14 134 GPFLGNIPK 6.000 15 497 VLFCLFIFV 6.000 16 318 GLGYLVPSL 5.400 17 117 RLLEGNFSL 4.050 18 178 CLGTRQCSR 4.000 19 105 SMEAQGLSF 4.000 20 136 FLGNIPKQY 3.000 21 69 WTYSGQWMY 3.000 22 9 LLQLTLTAF 3.000 23 13 TLTAFLTIL 2.700 24 404 QTLEAHQSK 2.250 25 26 HLLAPVFRT 2.025 26 447 YINYSEEIK 2.000 27 110 GLSFAQVRL 1.800 28 282 WLTGSNLTL 1.800 29 20 ILVQPQHLL 1.350 30 75 WMYERVWYP 1.350 31 496 IVLFCLFIF 1.350 32 518 SLNSQPLNL 1.200 33 434 QTACGTVGK 1.000 34 552 GLTTHQYDT 0.900 35 118 LLEGNFSLC 0.900 36 339 NLRSFIHKV 0.900 37 239 LLYQLFRNL 0.900 38 460 RLHEASENL 0.900 39 543 CQVSNRAMK 0.900 40 242 QLFRNLFCS 0.900 41 442 KQCCLYINY 0.720 42 289 TLSVNNSGL 0.600 43 359 NPPLYCNPK 0.600 44 343 FIHKVTPHR 0.600 45 329 YLTLNASQI 0.600 46 508 YVRVFRKSR 0.600 47 8 ALLQLTLTA 0.600 48 495 LIVLFCLFI 0.540 49 383 DLEKAILNI 0.540 50 228 CLYSCQNQT 0.500

[0402] TABLE X Scoring Results 103P2D6 HLA peptides A3 10-MERS Score (Estimate of Half Time of Disassociation of Start Subsequence a Molecule Containing Rank Position Residue Listing This Subsequence) 1 505 VLIYVRVFRK 270.000 2 228 CLYSCQNQTK 100.000 3 473 LLDWQGIFAK 90.000 4 394 AMEQEFSATK 60.000 5 242 QLFRNLFCSY 60.000 6 497 VLFCLFIFVL 40.500 7 460 RLHEASENLK 30.000 8 239 LLYQLFRNLF 30.000 9 252 GLTEAHGKWR 9.000 10 493 VLLIVLFCLF 9.000 11 445 CLYINYSEEI 9.000 12 337 ITNLRSFIHK 6.000 13 549 AMKGLTTHQY 6.000 14 494 LLIVLFCLFI 5.400 15 500 CLFIFVLIYV 4.500 16 8 ALLQLTLTAF 4.500 17 478 GIFAKVGDWF 4.500 18 11 QLTLTAFLTI 3.600 19 296 GLFFLCGNGV 3.000 20 142 KQYCNQILWF 2.700 21 178 CLGTRQCSRF 2.000 22 121 GNFSLCVENK 1.800 23 73 GQWMYERVWY 1.800 24 118 LLEGNFSLCV 1.800 25 3 SLSNCALLQL 1.800 26 403 KQTLEAHQSK 1.800 27 204 GLPNTQDYKW 1.800 28 495 LIVLFCLFIF 1.350 29 117 RLLEGNFSLC 1.350 30 438 GTVGKQCCLY 1.350 31 412 KVSSLASASR 1.200 32 499 FCLFIFVLIY 1.080 33 331 TLNASQITNL 0.900 34 304 GVYKGFPPKW 0.900 35 527 ALSPQQSAQL 0.900 36 504 FVLIYVRVFR 0.900 37 467 NLKNVPLLDW 0.900 38 424 HVLDIPTTQR 0.900 39 238 GLLYQLFRNL 0.810 40 75 WMYERVWYPQ 0.675 41 492 YVLLIVLFCL 0.608 42 49 HLDNAEQPEL 0.600 43 219 GLTWSGNDTC 0.600 44 246 NLFCSYGLTE 0.600 45 20 ILVQPQHLLA 0.600 46 9 LLQLTLTAFL 0.600 47 289 TLSVNNSGLF 0.600 48 37 ILTNQSNCWL 0.600 49 433 RQTACGTVGK 0.600 50 110 GLSFAQVRLL 0.540

[0403] TABLE XI Scoring Results 103P2D6 HLA peptides A11 9 MERS Score (Estimate of half Time of Disassociation of Start Subsequence a Molecule Containing Rank Position Residue Listing This Subsequence) 1 304 GVYKGFPPK 12.000 2 134 GPFLGNIPK 2.400 3 506 LIYVRVFRK 2.400 4 404 QTLEAHQSK 1.500 5 204 GLPNTQDYK 1.200 6 434 QTACGTVGK 1.000 7 543 CQVSNRAMK 0.900 8 378 SLGTYDLEK 0.800 9 320 GYLVPSLTR 0.720 10 229 LYSCQNQTK 0.400 11 447 YINYSEEIK 0.400 12 299 FLCGNGVYK 0.400 13 502 FIFVLIYVR 0.320 14 122 NFSLCVENK 0.200 15 508 YVRVFRKSR 0.200 16 359 NPPLYCNPK 0.200 17 338 TNLRSFIHK 0.120 18 474 LDWQGIFAK 0.120 19 156 GTFMPSIDV 0.120 20 505 VLIYVRVFR 0.120 21 208 TQDYKWVDR 0.120 22 308 GFPPKWSGR 0.120 23 79 RVWYPQAEV 0.120 24 253 LTEAHGKWR 0.100 25 496 IVLFCLFIF 0.090 26 178 CLGTRQCSR 0.080 27 425 VLDIPTTQR 0.080 28 343 FIHKVTPHR 0.080 29 161 SIDVTNESR 0.080 30 480 FAKVGDWFR 0.080 31 142 KQYCNQILW 0.072 32 540 ETSCQVSNR 0.060 33 395 MEQEFSATK 0.060 34 174 DTSVCLGTR 0.060 35 117 RLLEGNFSL 0.054 36 438 GTVGKQCCL 0.045 37 365 NPKDNSTIR 0.040 38 21 LVQPQHLLA 0.040 39 69 WTYSGQWMY 0.040 40 291 SVNNSGLFF 0.040 41 89 NHSTSSYRK 0.040 42 333 NASQITNLR 0.040 43 237 KGLLYQLFR 0.036 44 393 KAMEQEFSA 0.036 45 442 KQCCLYINY 0.036 46 251 YGLTEAHGK 0.030 47 414 SSLASASRK 0.030 48 12 LTLTAFLTI 0.030 49 337 ITNLRSFIH 0.030 50 380 GTYDLEKAI 0.030

[0404] TABLE XII Scoring Results 103P2D6 HLA peptides A11 10-MERS Score (Estimate of half Time of Disassociation of Start Subsequence a Molecule Containing Rank Position Residue Listing This Subsequence) 1 337 ITNLRSFIHK 2.000 2 505 VLIYVRVFRK 1.800 3 433 RQTACGTVGK 1.800 4 403 KQTLEAHQSK 1.800 5 473 LLDWQGIFAK 1.200 6 412 KVSSLASASR 1.200 7 460 RLHEASENLK 1.200 8 228 CLYSCQNQTK 0.800 9 504 FVLIYVRVFR 0.600 10 446 LYINYSEEIK 0.600 11 424 HVLDIPTTQR 0.600 12 508 YVRVFRKSRR 0.400 13 250 SYGLTEAHGK 0.400 14 394 AMEQEFSATK 0.400 15 298 FFLCGNGVYK 0.300 16 121 GNFSLCVENK 0.240 17 542 SCQVSNRAMK 0.200 18 262 CADASITNDK 0.200 19 207 NTQDYKWVDR 0.200 20 70 TYSGQWMYER 0.160 21 87 VQNHSTSSYR 0.120 22 304 GVYKGFPPKW 0.120 23 108 AQGLSFAQVR 0.120 24 384 LEKAILNISK 0.120 25 252 GLTEAHGKWR 0.120 26 501 LFIFVLIYVR 0.120 27 492 YVLLIVLFCL 0.090 28 479 IFAKVGDWFR 0.080 29 88 QNHSTSSYRK 0.080 30 142 KQYCNQILWF 0.072 31 193 RTWNSSAVPL 0.060 32 291 SVNNSGLFFL 0.060 33 97 KVTWHWEASM 0.060 34 380 GTYDLEKAIL 0.060 35 177 VCLGTRQCSR 0.060 36 496 IVLFCLFIFV 0.060 37 507 IYVRVFRKSR 0.060 38 342 SFIHKVTPHR 0.060 39 438 GTVGKQCCLY 0.045 40 439 TVGKQCCLYI 0.040 41 213 WVDRNSGLTW 0.040 42 133 SGPFLGNIPK 0.040 43 73 GQWMYERVWY 0.036 44 303 NGVYKGFPPK 0.030 45 203 IGLPNTQDYK 0.030 46 307 KGFPPKWSGR 0.024 47 478 GIFAKVGDWF 0.024 48 451 SEEIKSNIQR 0.024 49 204 GLPNTQDYKW 0.024 50 296 GLFFLCGNGV 0.024

[0405] TABLE XIII Scoring Results 103P2D6 HLA peptides A24 9-MERS Score (Estimate of half Time of Disassociation of Start Subsequence a Molecule Containing Rank Position Residue Listing This Subsequence) 1 381 TYDLEKAIL 200.000 2 491 GYVLLIVLF 180.000 3 240 LYQLFRNLF 180.000 4 143 QYCNQILWF 100.000 5 446 LYINYSEEI 82.500 6 398 EFSATKQTL 24.000 7 486 WFRSWGYVL 20.000 8 498 LFCLFIFVL 20.000 9 511 VFRKSRRSL 20.000 10 212 KWVDRNSGL 14.400 11 117 RLLEGNFSL 14.400 12 479 IFAKVGDWF 14.000 13 507 IYVRVFRKS 13.860 14 245 RNLFCSYGL 12.000 15 150 WFDSTDGTF 10.000 16 460 RLHEASENL 9.600 17 520 NSQPLNLAL 8.640 18 493 VLLIVLFCL 8.400 19 27 LLAPVFRTL 8.064 20 81 WYPQAEVQN 7.500 21 529 SPQQSAQLL 7.200 22 10 LQLTLTAFL 7.200 23 322 LVPSLTRYL 7.200 24 19 TILVQPQHL 7.200 25 94 SYRKVTWHW 7.000 26 210 DYKWVDRNS 7.000 27 38 LTNQSNCWL 6.000 28 449 NYSEEIKSN 6.000 29 20 ILVQPQHLL 6.000 30 545 VSNRAMKGL 6.000 31 4 LSNCALLQL 6.000 32 292 VNNSGLFFL 6.000 33 231 SCQNQTKGL 6.000 34 528 LSPQQSAQL 6.000 35 2 GSLSNCALL 6.000 36 518 SLNSQPLNL 6.000 37 438 GTVGKQCCL 6.000 38 232 CQNQTKGLL 6.000 39 466 ENLKNVPLL 6.000 40 194 TWNSSAVPL 6.000 41 371 TIRALFPSL 5.760 42 239 LLYQLFRNL 5.760 43 305 VYKGFPPKW 5.500 44 362 LYCNPKDNS 5.000 45 6 NCALLQLTL 4.800 46 235 QTKGLLYQL 4.800 47 42 SNCWLCEHL 4.800 48 103 EASMEAQGL 4.800 49 490 WGYVLLIVL 4.800 50 318 GLGYLVPSL 4.800

[0406] TABLE XIV Scoring Results 103P2D6 HLA peptides A24 10-MERS Score (Estimate of half Time of Disassociation of Start Subsequence a Molecule Containing Rank Position Residue Listing This Subsequence) 1 328 RYLTLNASQI 150.000 2 449 NYSEEIKSNI 84.000 3 375 LFPSLGTYDL 30.000 4 486 WFRSWGYVLL 20.000 5 503 IFVLIYVRVF 15.000 6 517 RSLNSQPLNL 12.000 7 491 GYVLLIVLFC 10.500 8 26 HLLAPVFRTL 10.080 9 370 STIRALFPSL 8.640 10 238 GLLYQLFRNL 8.640 11 321 YLVPSLTRYL 8.640 12 498 LFCLFIFVLI 8.400 13 492 YVLLIVLFCL 8.400 14 510 RVFRKSRRSL 8.000 15 193 RTWNSSAVPL 8.000 16 240 LYQLFRNLFC 7.500 17 320 GYLVPSLTRY 7.500 18 76 MYERVWYPQA 7.500 19 41 QSNCWLCEHL 7.200 20 18 LTILVQPQHL 7.200 21 528 LSPQQSAQLL 7.200 22 9 LLQLTLTAFL 7.200 23 317 CGLGYLVPSL 7.200 24 109 QGLSFAQVRL 6.000 25 29 APVFRTLSIL 6.000 26 331 TLNASQITNL 6.000 27 464 ASENLKNVPL 6.000 28 288 LTLSVNNSGL 6.000 29 231 SCQNQTKGLL 6.000 30 281 WWLTGSNLTL 6.000 31 362 LYGNPKDNST 6.000 32 291 SVNNSGLFFL 6.000 33 12 LTLTAFLTIL 6.000 34 19 TILVQPQHLL 6.000 35 381 TYDLEKAILN 5.000 36 305 VYKGFPPKWS 5.000 37 489 SWGYVLLIVL 4.800 38 234 NQTKGLLYQL 4.800 39 380 GTYDlEKAIL 4.800 40 519 LNSQpLNLAL 4.800 41 140 IPKQyCNQIL 4.800 42 5 SNCAlLQLTL 4.800 43 353 TQGDtDNPPL 4.800 44 527 ALSPqQSAQL 4.800 45 49 HLDNaEQPEL 4.400 46 493 VLLIvLFCLF 4.320 47 544 QVSNrAMKGL 4.000 48 323 VPSLtRYLTL 4.000 49 1 MGSLsNCALL 4.000 50 485 DWFRsWGYVL 4.000

[0407] TABLE XV Scoring Results 103P2D6 HLA peptides B7 9-MERS Score (Estimate of half Time of Disassociation of Start Subsequence a Molecule Containing Rank Position Residue Listing This Subsequence) 1 376 FPSLFTYDL 80.000 2 529 SPQQSAQLL 80.000 3 371 TIRALFPSL 40.000 4 314 SGRCGLGYL 40.00 5 29 APVFRTLSI 24.000 6 322 LVPSLTRYL 20.000 7 103 EASMEAQGL 12.000 8 418 SASRKDHVL 12.000 9 471 VPLLDWQGI 8.000 10 140 IPKQYCNQI 8.000 11 20 ILVQPQHLL 6.000 12 197 SSAVPLIGL 6.000 13 275 GHRTPTWWL 6.000 14 511 VFRKSRRSL 6.000 15 235 QTKGLLYQL 4.000 16 453 EIKSNIQRL 4.000 17 110 GLSFAQVRL 4.000 18 493 VLLIVLFCL 4.000 19 486 WFRSWGYVL 4.000 20 282 WLTGSNLTL 4.000 21 4 LSNCALLQL 4.000 22 545 VSNRAMKGL 4.000 23 520 NSQPLNLAL 4.000 24 42 SNCWLCEHL 4.000 25 231 SCQNQTKGL 4.000 26 239 LLYQLFRNL 4.000 27 528 LSPQQSAQL 4.000 28 13 TLTAFLTIL 4.000 29 10 LQLTLTAFL 4.000 30 490 WGYVLLIVL 4.000 31 27 LLAPVFRTL 4.000 32 111 LSFAQVRLL 4.000 33 466 ENLKNVPLL 4.000 34 518 SLNSQPLNL 4.000 35 2 GSLSNCALL 4.000 36 19 TILVQPQHL 4.000 37 6 NCALLQLTL 4.000 38 318 GLGYLVPSL 4.000 39 332 LNASQITNL 4.000 40 460 RLHEASENL 4.000 41 1 MGSLSNCAL 4.000 42 289 TLSVNNSGL 4.000 43 23 QPQHLLAPV 4.000 44 438 GTVGKQCCL 4.000 45 232 CQNQTKGLL 4.000 46 245 RNLFCSYGL 4.000 47 38 LTNQSNCWL 4.000 48 117 RLLEGNFSL 4.000 49 292 VNNSGLFFL 4.000 50 554 TTHQYDTSL 4.000

[0408] TABLE XVI Scoring Results 103P2D6 HLA peptides B7 10-MERS Score (Estimate of half Time of Disassociation of Start Subsequence a Molecule Containing Rank Position Residue Listing This Subsequence) 1 29 APVFRTLSIL 240.000 2 140 IPKQYCNQIL 80.000 3 323 VPSLTRYLTL 80.000 4 510 RVFRKSRRSL 30.000 5 492 YVLLIVLFCL 20.000 6 291 SVNNSGLFFL 20.000 7 544 QVSNRAMKGL 20.000 8 310 PPKWSGRCGL 12.000 9 417 ASASRKDHVL 12.000 10 527 ALSPQQSAQL 12.000 11 407 EAHQSKVSSL 12.000 12 419 ASRKDHVLDI 12.000 13 196 NSSAVPLIGL 6.000 14 274 DGHRTPTWWL 6.000 15 19 TILVQPQHLL 6.000 16 97 KVTWHWEASM 5.000 17 109 QGLSFAQVRL 4.000 18 220 LTWSGNDTCL 4.000 19 528 LSPQQSAQLL 4.000 20 331 TLNASQITNL 4.000 21 370 STIRALFPSL 4.000 22 437 CGTVGKQCCL 4.000 23 110 GLSFAQVRLL 4.000 24 231 SCQNQTKGLL 4.000 25 353 TQGDTDNPPL 4.000 26 193 RTWNSSAVPL 4.000 27 517 RSLNSQPLNL 4.000 28 41 QSNCWLCEHL 4.000 29 519 LNSQPLNLAL 4.000 30 238 GLLYQLFRNL 4.000 31 26 HLLAPVFRTL 4.000 32 234 NQTKGLLYQL 4.000 33 205 LPNTQDYKWV 4.000 34 128 ENKNGSGPFL 4.000 35 486 WFRSWGYVLL 4.000 36 12 LTLTAFLTIL 4.000 37 529 SPQQSAQLLV 4.000 38 553 LTTHQYDTSL 4.000 39 317 CGLGYLVPSL 4.000 40 288 LTLSVNNSGL 4.000 41 380 GTYDLEKAIL 4.000 42 18 LTILVQPQHL 4.000 43 554 TTHQYDTSLL 4.000 44 37 ILTNQSNCWL 4.000 45 515 SRRSLNSQPL 4.000 46 9 LLQLTLTAFL 4.000 47 321 YLVPSLTRYL 4.000 48 497 VLFCLFIFVL 4.000 49 5 SNCALLQLTL 4.000 50 3 SLSNCALLQL 4.000

[0409] TABLE XVII Scoring Results 103P2D6 HLA peptides B35 9-MERS Score (Estimate of half Time of Disassociation of Start Subsequence a Molecule Containing Rank Position Residue Listing This Subsequence) 1 140 IPKQYCNQI 24.000 2 376 FPSLGTYDL 20.000 3 529 SPQQSAQLL 20.000 4 222 WSGNDTCLY 15.000 5 391 ISKAMEQEF 15.000 6 471 VPLLDWQGI 12.000 7 63 ASASTWWTY 10.000 8 313 WSGRCGLGY 10.000 9 61 VPASASTWW 10.000 10 205 LPNTQDYKW 10.000 11 29 APVFRTLSI 8.000 12 528 LSPQQSAQL 5.000 13 520 NSQPLNLAL 5.000 14 545 VSNRAMKGL 5.000 15 197 SSAVPLIGL 5.000 16 111 LSFAQVRLL 5.000 17 2 GSLSNCALL 5.000 18 290 LSVNNSGLF 5.000 19 4 LSNCALLQL 5.000 20 103 EASMEAQGL 4.500 21 23 QPQHLLAPV 4.000 22 117 RLLEGNFSL 4.000 23 460 RLHEASENL 4.000 24 442 KQCCLYINY 4.000 25 488 RSWGYVLLI 4.000 26 314 SGRCGLGYL 3.000 27 235 QTKGLLYQL 3.000 28 453 EIKSNIQRL 3.000 29 418 SASRKDHVL 3.000 30 128 ENKNGSGPF 3.000 31 371 TIRALFPSL 3.000 32 115 QVRLLEGNF 3.000 33 92 TSSYRKVTW 2.500 34 167 ESRNDDDDT 2.250 35 69 WTYSGQWMY 2.000 36 482 KVGDWFRSW 2.000 37 200 VPLIGLPNT 2.000 38 309 FPPKWSGRC 2.000 39 323 VPSLTRYLT 2.000 40 203 IGLPNTQDY 2.000 41 136 FLGNIPKQY 2.000 42 500 CLFIFVLIY 2.000 43 387 AILNISKAM 2.000 44 245 RNLFCSYGL 2.000 45 439 TVGKQCCLY 2.000 46 321 YLVPSLTRY 2.000 47 428 IPTTQRQTA 2.000 48 132 GSGPFLGNI 2.000 49 98 VTWHWEASM 2.000 50 278 TPTWWLTGS 2.000

[0410] TABLE XVIII Scoring Results 103P2D6 HLA peptides A35 10-MERS Score (Estimate of half Time of Disassociation of Start Subsequence a Molecule Containing Rank Position Residue Listing This Subsequence) 1 140 IPKQYCNQIL 60.000 2 323 VPSLTRYLTL 20.000 3 23 QPQHLLAPVF 20.000 4 471 VPLLDWQGIF 20.000 5 29 APVFRTLSIL 20.000 6 365 NPKDNSTIRA 12.000 7 386 KAILNISKAM 12.000 8 373 RALFPSLGTY 12.000 9 517 RSLNSQPLNL 10.000 10 104 ASMEAQGLSF 10.000 11 541 TSCQVSNRAM 10.000 12 549 AMKGLTTHQY 6.000 13 310 PPKWSGRCGL 6.000 14 205 LPNTQDYKWV 6.000 15 419 ASRKDHVLDI 6.000 16 528 LSPQQSAQLL 5.000 17 230 YSCQNQTKGL 5.000 18 334 ASQITNLRSF 5.000 19 313 WSGRCGLGYL 5.000 20 41 QSNCWLCEHL 5.000 21 417 ASASRKDHVL 5.000 22 196 NSSAVPLIGL 5.000 23 290 LSVNNSGLFF 5.000 24 529 SPQQSAQLLV 4.000 25 97 KVTWHWEASM 4.000 26 353 TQGDTDNPPL 3.000 27 235 QTKGLLYQLF 3.000 28 128 ENKNGSGPFL 3.000 29 380 GTYDLEKAIL 3.000 30 407 EAHQSKVSSL 3.000 31 73 GQWMYERVWY 3.000 32 93 SSYRKVTWHW 2.500 33 391 ISKAMEQEFS 2.250 34 167 ESRNDDDDTS 2.250 35 438 GTVGKQCCLY 2.000 36 82 YPQAEVQNHS 2.000 37 232 CQNQTKGLLY 2.000 38 242 QLFRNLFCSY 2.000 39 61 VPASASTWWT 2.000 40 510 RVFRKSRRSL 2.000 41 51 DNAEQPELVF 2.000 42 159 MPSIDVTNES 2.000 43 343 TPHRCTQGDT 2.000 44 488 RSWGYVLLIV 2.000 45 278 TPTWWLTGSN 2.000 46 142 KQYCNQILWF 2.000 47 193 RTWNSSAVPL 2.000 48 86 EVQNHSTSSY 2.000 49 202 LIGLPNTQDY 2.000 50 499 FCLFIFVLIY 2.000

[0411] TABLE XIX Motif-bearing Subsequences of the 103P2D6 Protein Post-translational modification Sites 15 N-glycosylation sites 1  40-43 NQSN 2  89-92 NHST 3 122-125 NFSL 4 131-134 NGSG 5 166-169 NESR 6 192-195 NRTW 7 196-199 NSSA 8 225-228 NDTC 9 234-237 NQTK 10 287-290 NLTL 11 293-296 NNSG 12 333-336 NASQ 13 369-372 NSTI 14 390-393 NISK 15 449-452 NYSE One cAMP- and cGMP-dependent protein kinase phosphorylation site 1  96-99 RKVT Eight protein kinase C phosphorylation sites 1  94-96 SYR 2 191-193 TNR 3 314-316 SGR 4 371-373 TIR 5 420-422 SRK 6 431-433 TQR 7 515-517 SRR 8 546-548 SNR Signicance of Post translational modications Four casein kinase II phosphorylation site 1 168-171 SRND 2 223-226 SGND 3 353-356 TQGD 4 420-423 SRKD Nine N-myristoylation sites 1  2-7 GSLSNC 2 110-115 GLSFAQ 3 180-185 GTRQCS 4 204-209 GLPNTQ 5 219-224 GLTWSG 6 224-229 GNDTCL 7 252-257 GLTEAH 8 285-290 GSNLTL 9 304-309 GVYKGF

[0412] Glycosylation, or attachment of carbohydrate chains to asparagines, serine or threonine moieties, plays an important role in protein folding, stability and protection from degradation (Biochem J. 2000, 348:1). In addition, glycosylation allows sorting of proteins from the endoplasmic reticulum along the secretory pathway, thereby contributing to protein localization (FEBS Lett. 2000, 476:32). Also, glycosylation often contributes to cell adhesion and immune recognition (J. Mol. Biol. 1999, 293:351).

[0413] Phosphorylation, whether by PKC, cAMP and c-GMP dependent kinases, or casein kinases, exhorts a profound effect on proteins. Phosphorylation mediates protein-protein interactions as well as signaling pathway activation. Phosphorylation also controls protein localization, translocation and enzymatic activity, and regulates transcriptional activation (Mol Immunol. 2000, 37: 1; Cell Mol Life Sci. 2000, 57:1172-83). By means of post-translational modification of protein, phosphorylation regulates cellular functions, including proliferation, migration and gene expression (Cell Prolif. 2000, 33:341; Mol Biol Cell. 2001, 12:351).

[0414] Myristoylation serves to anchor numerous proteins to the cytoplasmic face of the plasma membrane. This process serves to facilitate protein recruitment, complex assembly and signaling through proteins (Current Opinion Cell Biol. 1994, 6:219; J. Biol. Chem. 1996, 271:1573).

[0415] Motifs found in 103P2D6

[0416] 14-35 Leucine zipper pattern LTAFLTILVQPQHLLAPVFRTL

[0417] 484-516 Large-conductance mechanosensitive channel, GDWFRSWGYVLLIVLFCLFIFVLIYVRVFRKSRR

[0418] 487-507 Sodium/chloride neurotransmitter symporter signature FRSWGYVLLIVLFCLFIFVLI

[0419] Topology and Transmembrane Domains

[0420] Using three different prediction programs, 103P2D6 is proposed to be a membrane associated protein, primarily expressed at the cell surface (64%). There is a possibility that 103P2D6 is associated with the endoplasmic reticulum (21%).

[0421] Several possibilities for transmembrane domains and topologies:

[0422] PSORT (http://psort.nibb.ac.jp) indicates the presence of 1 TM domain at aa 493-509 (VLLIVLFCLFIFVLIYV), and 1 signal sequence at aa 1-24 (MGSLSNCALLQLTLTAFLTILVQP), with a cleavage site between aa 24 and aa 25.

[0423] TMpred (www.ch.3 mbnet.org) indicates that 103P2D6 may have four transmembrane domains, which are listed below:

[0424] TM1 aa 4-22-o-i

[0425] TM2 aa 58-77-i-o

[0426] TM3 aa 283-300-o-i

[0427] TM4 aa 493-509-i-o

[0428]  Although this scenario is less likely to occur, in view of the similarity with envelope protein which has a single transmembrane domain, it is possible that 103P2D6 exhibits a multiple TM configuration

[0429] In all cases, the N-terminus extends outside the cell, while the C-terminus is intracellular. TABLE XX Frequently Occurring Motifs avg. % Name identity Description Potential Function zf-C2H2 34% Zinc finger, C2H2 type Nucleic acid-binding protein functions as tanscription factor, nuclear location probable cytochrome b N 68% Cytochrome b(N- membrane bound oxidase, generate terminal)/b6/petB superoxide ig 19% Immunoglobulin domain domains are one hundred amino acids long and include a conserved intradomain disulfide bond. WD40 18% WD domain, G-beta repeat tandem repeats of about 40 residues, each containing a Trp-Asp motif. Function in signal transduction and protein interaction PDZ 23% PDZ domain may function in targeting signaling molecules to sub-membranous sites LRR 28% Leucine Rich Repeat short sequence motifs involved in protein- protein interactions pkinase 23% Protein kinase domain conserved catalytic core common to both serine/threonine and tyrosine protein kinases containing an ATP binding site and a catalytic site PH 16% PH domain pleckstrin homology involved in intracellular signaling or as constituents of the cytoskeleton EGF 34% EGF-like domain 30-40 amino-acid long found in the extracellular domain of membrane-bound proteins or in secreted proteins rvt 49% Reverse transcriptase (RNA- dependent DNA polymerase) ank 25% Ank repeat Cytoplasmic protein, associates integral membrane proteins to the cytoskeleton oxidored q1 32% NADH- membrane associated. Involved in proton Ubiquinone/plastoquinone translocation across the membrane (complex I), various chains efhand 24% EF hand calcium-binding domain, consists of a 12 residue loop flanked on both side by a 12 residue alpha-helical domain rvp 79% Retroviral aspartyl protease Aspartyl or acid proteases, centered on a catalytic aspartyl residue Collagen 42% Collagen triple helix repeat extracellular structural proteins involved in 20 copies) formation of connective tissue. The sequence consists of the G-X-Y and the polypeptide chains forms a triple helix. fn3 20% Fibronectin type III domain Located in the extracellular ligand-binding region of receptors and is about 200 amino acid residues long with two pairs of cysteines involved in disulfide bonds 7tm 1 19% 7 transmembrane receptor seven hydrophobic transmembrane (rhodopsin family) regions, with the N-terminus located extracellularly while the C-terminus is cytoplasmic. Signal through G proteins

[0430]

1 752 1 4728 DNA homo sapiens CDS (805)...(2496) 1 cacctgtgac tgttgatgtg gaactgattt atcgcgtatt cgtactggct gaatccggct 60 gtccgctctg cggtgccccg ccccgcccca acccaggatc ttcccagccc cgctccgccc 120 caacccagga tcttcccagc cctcgtgtgt ccccgctcac ttcagttccc gccgcgagcc 180 ttcttcttgg tcttctggcc tggcggcgat cgtcggccag tttatccctc ggagttgcac 240 tggcagacac gccgctactt tgtagcgggt ttcgggcggg ccacgcgtgc ggcgacagga 300 acccaacccc ggccgacctt gggctccagg aattcgttgt ctacgtctgc ggaggtgcgg 360 cagcctcagt tttaagcgca ggtgatcaag tatatctgaa agtttttaga agaaaagatg 420 tgttgtaacc tccctggaaa aagaagagac cttatgaagt actgctaacc acctacatag 480 tcatcaaaat aaaattcctg aatttgtaca agccacagga agctagattg agatcattat 540 atgacaactg gaaggccaag gctatgggtt acctcaaatt gaggaatttc ggcacctact 600 cacaggctcc atgagcagat gaagtagaca gctttactca gtatctcaga ccaagaactt 660 catctccatc tccaactagc tgaaacatct tccctcctca acctggaaaa ttctctgact 720 tagaaattta aacaaaaccc tcccctttca ttgaatctcc attgtctgga gtttgcttgt 780 tttaatctag gctgttcctc cact atg ggc tcc ctt tca aac tgt gcc ctg 831 Met Gly Ser Leu Ser Asn Cys Ala Leu 1 5 ctt caa cta acc ctt act gct ttt ttg aca att cta gta caa cct cag 879 Leu Gln Leu Thr Leu Thr Ala Phe Leu Thr Ile Leu Val Gln Pro Gln 10 15 20 25 cac ctg ctt gct cca gtt ttc cgg aca cta tct atc ttg act aat cag 927 His Leu Leu Ala Pro Val Phe Arg Thr Leu Ser Ile Leu Thr Asn Gln 30 35 40 tct aat tgc tgg tta tgt gaa cat cta gat aat gca gaa caa ccc gaa 975 Ser Asn Cys Trp Leu Cys Glu His Leu Asp Asn Ala Glu Gln Pro Glu 45 50 55 cta gtt ttt gtt cct gcc agt gca agc acc tgg tgg acc tat tct gga 1023 Leu Val Phe Val Pro Ala Ser Ala Ser Thr Trp Trp Thr Tyr Ser Gly 60 65 70 caa tgg atg tat gaa agg gtg tgg tat cca caa gca gaa gta cag aat 1071 Gln Trp Met Tyr Glu Arg Val Trp Tyr Pro Gln Ala Glu Val Gln Asn 75 80 85 cac tct act tcc tcc tat cgt aaa gtg act tgg cac tgg gaa gcc tcc 1119 His Ser Thr Ser Ser Tyr Arg Lys Val Thr Trp His Trp Glu Ala Ser 90 95 100 105 atg gaa gct caa ggt cta tcc ttt gct caa gta agg tta ttg gag gga 1167 Met Glu Ala Gln Gly Leu Ser Phe Ala Gln Val Arg Leu Leu Glu Gly 110 115 120 aat ttt tct ctt tgc gta gaa aat aaa aat ggc agt gga ccc ttc cta 1215 Asn Phe Ser Leu Cys Val Glu Asn Lys Asn Gly Ser Gly Pro Phe Leu 125 130 135 ggt aat ata cct aaa caa tac tgt aat caa ata cta tgg ttt gat tct 1263 Gly Asn Ile Pro Lys Gln Tyr Cys Asn Gln Ile Leu Trp Phe Asp Ser 140 145 150 aca gat ggc acc ttc atg ccc tct ata gat gtt aca aat gaa tcc agg 1311 Thr Asp Gly Thr Phe Met Pro Ser Ile Asp Val Thr Asn Glu Ser Arg 155 160 165 aac gat gat gat gat aca agt gtt tgc cta ggc act aga caa tgt tcc 1359 Asn Asp Asp Asp Asp Thr Ser Val Cys Leu Gly Thr Arg Gln Cys Ser 170 175 180 185 cgg ttt gca ggt tgc aca aac cgg acc tgg aac agc tca gct gtt ccc 1407 Arg Phe Ala Gly Cys Thr Asn Arg Thr Trp Asn Ser Ser Ala Val Pro 190 195 200 ttg att ggt ctg ccc aat acc caa gac tac aaa tgg gta gat cga aat 1455 Leu Ile Gly Leu Pro Asn Thr Gln Asp Tyr Lys Trp Val Asp Arg Asn 205 210 215 tct gga ttg acc tgg tca ggt aat gac acc tgt ctc tat agc tgc caa 1503 Ser Gly Leu Thr Trp Ser Gly Asn Asp Thr Cys Leu Tyr Ser Cys Gln 220 225 230 aac caa acc aaa ggc ctt ctg tac cag cta ttt cgc aac cta ttt tgc 1551 Asn Gln Thr Lys Gly Leu Leu Tyr Gln Leu Phe Arg Asn Leu Phe Cys 235 240 245 tct tat ggc ctg aca gag gca cat ggg aaa tgg aga tgt gca gat gcc 1599 Ser Tyr Gly Leu Thr Glu Ala His Gly Lys Trp Arg Cys Ala Asp Ala 250 255 260 265 agc ata act aat gac aaa ggt cat gat gga cac cgg acc ccc acc tgg 1647 Ser Ile Thr Asn Asp Lys Gly His Asp Gly His Arg Thr Pro Thr Trp 270 275 280 tgg ctc aca ggt tcc aat ctg acc ttg tct gtg aac aac tct ggc ctc 1695 Trp Leu Thr Gly Ser Asn Leu Thr Leu Ser Val Asn Asn Ser Gly Leu 285 290 295 ttt ttt ttg tgc ggc aat ggg gtg tac aaa ggg ttt cca cct aaa tgg 1743 Phe Phe Leu Cys Gly Asn Gly Val Tyr Lys Gly Phe Pro Pro Lys Trp 300 305 310 tct ggg cga tgt gga ctt ggg tat ctt gta cct tcc ctc acc aga tac 1791 Ser Gly Arg Cys Gly Leu Gly Tyr Leu Val Pro Ser Leu Thr Arg Tyr 315 320 325 ctc acc tta aat gct agc caa att aca aac ctg aga tcc ttc att cat 1839 Leu Thr Leu Asn Ala Ser Gln Ile Thr Asn Leu Arg Ser Phe Ile His 330 335 340 345 aaa gta aca ccg cat aga tgc acc caa gga gac aca gac aat cca cct 1887 Lys Val Thr Pro His Arg Cys Thr Gln Gly Asp Thr Asp Asn Pro Pro 350 355 360 ctg tat tgc aac ccc aag gac aat tca aca ata agg gcc ctt ttt cca 1935 Leu Tyr Cys Asn Pro Lys Asp Asn Ser Thr Ile Arg Ala Leu Phe Pro 365 370 375 agt ttg gga act tat gat tta gaa aag gca att cta aac att tcc aaa 1983 Ser Leu Gly Thr Tyr Asp Leu Glu Lys Ala Ile Leu Asn Ile Ser Lys 380 385 390 gca atg gaa cag gaa ttc agt gcc act aag cag acc ttg gaa gca cac 2031 Ala Met Glu Gln Glu Phe Ser Ala Thr Lys Gln Thr Leu Glu Ala His 395 400 405 caa tca aaa gtt agc agt tta gcc tct gca tcc cga aag gat cat gtc 2079 Gln Ser Lys Val Ser Ser Leu Ala Ser Ala Ser Arg Lys Asp His Val 410 415 420 425 ttg gat ata ccg acc acc caa cga caa acg gct tgt gga act gtt ggc 2127 Leu Asp Ile Pro Thr Thr Gln Arg Gln Thr Ala Cys Gly Thr Val Gly 430 435 440 aaa cag tgt tgc ctc tat ata aat tat tcg gaa gaa ata aag tct aat 2175 Lys Gln Cys Cys Leu Tyr Ile Asn Tyr Ser Glu Glu Ile Lys Ser Asn 445 450 455 ata cag cgt ctc cac gaa gca tcc gag aac ctg aag aat gta cca tta 2223 Ile Gln Arg Leu His Glu Ala Ser Glu Asn Leu Lys Asn Val Pro Leu 460 465 470 ctt gat tgg caa ggc ata ttt gca aaa gtg gga gac tgg ttc aga tca 2271 Leu Asp Trp Gln Gly Ile Phe Ala Lys Val Gly Asp Trp Phe Arg Ser 475 480 485 tgg ggc tat gtg ctt tta att gtt ctt ttc tgc tta ttc atc ttt gtt 2319 Trp Gly Tyr Val Leu Leu Ile Val Leu Phe Cys Leu Phe Ile Phe Val 490 495 500 505 tta atc tat gtt cgt gtc ttt cgc aaa tct cgc aga tcc ctt aac tcc 2367 Leu Ile Tyr Val Arg Val Phe Arg Lys Ser Arg Arg Ser Leu Asn Ser 510 515 520 caa cct ctg aac cta gcc tta tct cca cag caa tca gca cag ctc ctt 2415 Gln Pro Leu Asn Leu Ala Leu Ser Pro Gln Gln Ser Ala Gln Leu Leu 525 530 535 gtc agt gaa act tca tgt caa gtt tca aat agg gca atg aag gga cta 2463 Val Ser Glu Thr Ser Cys Gln Val Ser Asn Arg Ala Met Lys Gly Leu 540 545 550 aca acc cat caa tat gac aca agt cta ctt tga gaatatctga acaaacagca 2516 Thr Thr His Gln Tyr Asp Thr Ser Leu Leu * 555 560 gctgcagaca aaaagcctta gctaaacttt gatgagtaaa gcaggtctta ccgagaattc 2576 agctgccaaa accctcctct gagtgttcct cttataaggg cacttagcac taggacctcc 2636 caaggtattg taaataagcc ttatcagaac tttttgtagt ttcattctga agccttaaga 2696 cacacaccat aaagctgatc tgtaaaacct taccccttgc tgttcagaga gctactcttt 2756 gtagtgttct tgcatgcata tataataaat gttttttcta ttgatctgtt aatttgcaag 2816 cccccaaaca ctggaactaa gttgggggca ggatagtttc tcccaacagc actttgtagg 2876 cttctggata gaccaaagag tgtgtttgaa tagataaggg aattttgttc ccttgatttt 2936 ggttgaaggt agaagaatcc aatgtacaca tacacaaaac tatgtcctta aatttgtctc 2996 aaagaataaa tattgggtag tcatcagaat tgactgaaaa cttactttag ggagaaagcc 3056 acaaataatt taggctggta tgatctgagc tttcctgatt tcccatgggc attttagtgg 3116 tctaatggaa tattagtctg atatgcattt tagccatctt atgaaatggg ccgcaggtgg 3176 aagtaggatg cagggagtgt tgaggtggca tctattatat caaatgcttc actctgccca 3236 gtcctctgtg agttccttac atgcatcttt ctagttaatc ctctcggtgt tcatatttac 3296 agattaatca actcaaaggg tagatagctt gctaaagatt atactattac tgaaaaatag 3356 caagatgcag aacagcatac ctagtatgtt actctttttt tttttttttt ttgaggcaga 3416 gtctggccct ttcacccagg ctggagtgca gtggctcaat ctcggctcac tgcaacctct 3476 gcccccttgg gttcatgcca ttctcctgcc tcccagccac tcaggaggct gaggcaggag 3536 gatcgcttga acccaggaag cggaggttgc ggtgagctga gatcgcacca ctgtattcca 3596 gcctggcaac agagtgagat tctgtggcaa aaaaaaaaaa aaaaaaaagc acagactggg 3656 tgtggtggct catgcctgta attccagcac tttgcgaggc tcaagcgatc ctttggcctc 3716 ggcctcccaa agtgcatgag ccaccatgcc tggcctgttt agttttgttt caagttgaaa 3776 tacctttctt gtgttttcta attagaaaag taatatctac tcattgtaaa aactcaaaca 3836 gtgcagaaat gtagaaagta gaaagtgtaa gtccctggtt gtcccttctg cctgagacaa 3896 ccactgctca cagtttgatg tatatccttc cagagactct caaatttaag caaataattt 3956 ttattaccat gtctttttat ttgaagacgt tacatttgcc tccaaagttc aacacaagtt 4016 caactgacca tatccttcca tgacctgaat agatgctatc ctttatcacg atgttcaatt 4076 gcctttgaaa gagagtagtc caggtatatt cctgatcaaa atttggcatt tttgatgata 4136 ctactctaca cagatcagac tcatgtgcag aatcgtgcct gaagagagag gtttggttaa 4196 gacagagatt tctggaaaca ttcaaattgc aaatggaaac ttgaaaccca caatctaatg 4256 aggaatgtac tggaaaaata atctgaagag ttgacaaatt gtgtactaga ttgaacacat 4316 ggaatgcaat gcaatgagac tttctgcact aaaacttatc ctcatatgta caacaatgat 4376 gtgtgtatta tataacagtg atgtgtacat ttctgacacc ccatacataa tatacacagt 4436 ttgtataaat gcatacattt aaaaatatat atgtacaata cagctaacat aaaactgtag 4496 tacgcctgaa ggatattact agtgcctaat attgagtatg agtcactgcg tgttcgcatc 4556 aacttggaag tgcagtaatt gttataaaat taatcagtgc agccaacatt atttatgaat 4616 cacatctttg aaactgtgca gtagcatata catatatatt tttaaataac atttttcaca 4676 gttttccaga gttactgttg aaatctgcat caccaaaaaa aaaaaaaaaa aa 4728 2 563 PRT homo sapiens 2 Met Gly Ser Leu Ser Asn Cys Ala Leu Leu Gln Leu Thr Leu Thr Ala 1 5 10 15 Phe Leu Thr Ile Leu Val Gln Pro Gln His Leu Leu Ala Pro Val Phe 20 25 30 Arg Thr Leu Ser Ile Leu Thr Asn Gln Ser Asn Cys Trp Leu Cys Glu 35 40 45 His Leu Asp Asn Ala Glu Gln Pro Glu Leu Val Phe Val Pro Ala Ser 50 55 60 Ala Ser Thr Trp Trp Thr Tyr Ser Gly Gln Trp Met Tyr Glu Arg Val 65 70 75 80 Trp Tyr Pro Gln Ala Glu Val Gln Asn His Ser Thr Ser Ser Tyr Arg 85 90 95 Lys Val Thr Trp His Trp Glu Ala Ser Met Glu Ala Gln Gly Leu Ser 100 105 110 Phe Ala Gln Val Arg Leu Leu Glu Gly Asn Phe Ser Leu Cys Val Glu 115 120 125 Asn Lys Asn Gly Ser Gly Pro Phe Leu Gly Asn Ile Pro Lys Gln Tyr 130 135 140 Cys Asn Gln Ile Leu Trp Phe Asp Ser Thr Asp Gly Thr Phe Met Pro 145 150 155 160 Ser Ile Asp Val Thr Asn Glu Ser Arg Asn Asp Asp Asp Asp Thr Ser 165 170 175 Val Cys Leu Gly Thr Arg Gln Cys Ser Arg Phe Ala Gly Cys Thr Asn 180 185 190 Arg Thr Trp Asn Ser Ser Ala Val Pro Leu Ile Gly Leu Pro Asn Thr 195 200 205 Gln Asp Tyr Lys Trp Val Asp Arg Asn Ser Gly Leu Thr Trp Ser Gly 210 215 220 Asn Asp Thr Cys Leu Tyr Ser Cys Gln Asn Gln Thr Lys Gly Leu Leu 225 230 235 240 Tyr Gln Leu Phe Arg Asn Leu Phe Cys Ser Tyr Gly Leu Thr Glu Ala 245 250 255 His Gly Lys Trp Arg Cys Ala Asp Ala Ser Ile Thr Asn Asp Lys Gly 260 265 270 His Asp Gly His Arg Thr Pro Thr Trp Trp Leu Thr Gly Ser Asn Leu 275 280 285 Thr Leu Ser Val Asn Asn Ser Gly Leu Phe Phe Leu Cys Gly Asn Gly 290 295 300 Val Tyr Lys Gly Phe Pro Pro Lys Trp Ser Gly Arg Cys Gly Leu Gly 305 310 315 320 Tyr Leu Val Pro Ser Leu Thr Arg Tyr Leu Thr Leu Asn Ala Ser Gln 325 330 335 Ile Thr Asn Leu Arg Ser Phe Ile His Lys Val Thr Pro His Arg Cys 340 345 350 Thr Gln Gly Asp Thr Asp Asn Pro Pro Leu Tyr Cys Asn Pro Lys Asp 355 360 365 Asn Ser Thr Ile Arg Ala Leu Phe Pro Ser Leu Gly Thr Tyr Asp Leu 370 375 380 Glu Lys Ala Ile Leu Asn Ile Ser Lys Ala Met Glu Gln Glu Phe Ser 385 390 395 400 Ala Thr Lys Gln Thr Leu Glu Ala His Gln Ser Lys Val Ser Ser Leu 405 410 415 Ala Ser Ala Ser Arg Lys Asp His Val Leu Asp Ile Pro Thr Thr Gln 420 425 430 Arg Gln Thr Ala Cys Gly Thr Val Gly Lys Gln Cys Cys Leu Tyr Ile 435 440 445 Asn Tyr Ser Glu Glu Ile Lys Ser Asn Ile Gln Arg Leu His Glu Ala 450 455 460 Ser Glu Asn Leu Lys Asn Val Pro Leu Leu Asp Trp Gln Gly Ile Phe 465 470 475 480 Ala Lys Val Gly Asp Trp Phe Arg Ser Trp Gly Tyr Val Leu Leu Ile 485 490 495 Val Leu Phe Cys Leu Phe Ile Phe Val Leu Ile Tyr Val Arg Val Phe 500 505 510 Arg Lys Ser Arg Arg Ser Leu Asn Ser Gln Pro Leu Asn Leu Ala Leu 515 520 525 Ser Pro Gln Gln Ser Ala Gln Leu Leu Val Ser Glu Thr Ser Cys Gln 530 535 540 Val Ser Asn Arg Ala Met Lys Gly Leu Thr Thr His Gln Tyr Asp Thr 545 550 555 560 Ser Leu Leu 3 342 DNA homo sapiens 3 gatcagcttt atggtgtgtg tcttaaggct tcagaatgaa actacaaaaa gttctgataa 60 ggcttattta caataccttg ggaggtccta gtgctaagtg cccttataag aggaacactc 120 agaggagggt tttggcagct gaattctcgg taagacctgc tttactcatc aaagtttagc 180 taaggctttt tgtctgcagc tgctgtttgt tcagatattc tcaaagtaga cttgtgtcat 240 attgatgggt tgttagtccc ttcattgccc tatttgaaac ttgacatgaa gtttcactga 300 caaggagctg tgctgattgc tgtggagata aggctaggtt ca 342 4 576 PRT homo sapiens 4 Ser Asn Thr Ser Thr Leu Met Lys Phe Tyr Ser Leu Leu Leu Tyr Ser 1 5 10 15 Leu Leu Phe Ser Phe Pro Phe Leu Cys His Pro Leu Pro Leu Pro Ser 20 25 30 Tyr Leu His His Thr Ile Asn Leu Thr His Ser Leu Leu Ala Ala Ser 35 40 45 Asn Pro Ser Leu Val Asn Asn Cys Trp Leu Cys Ile Ser Leu Ser Ser 50 55 60 Ser Ala Tyr Thr Ala Val Pro Ala Val Gln Thr Asp Trp Ala Thr Ser 65 70 75 80 Pro Ile Ser Leu His Leu Arg Thr Ser Phe Asn Ser Pro His Leu Tyr 85 90 95 Pro Pro Glu Glu Leu Ile Tyr Phe Leu Asp Arg Ser Ser Lys Thr Ser 100 105 110 Pro Asp Ile Ser His Gln Gln Ala Ala Ala Leu Leu Arg Thr Tyr Leu 115 120 125 Lys Asn Leu Ser Pro Tyr Ile Asn Ser Thr Pro Pro Ile Phe Gly Pro 130 135 140 Leu Thr Thr Gln Thr Thr Ile Pro Val Ala Ala Pro Leu Cys Ile Ser 145 150 155 160 Trp Gln Arg Pro Thr Gly Ile Pro Leu Gly Asn Leu Ser Pro Ser Arg 165 170 175 Cys Ser Phe Thr Leu His Leu Arg Ser Pro Thr Thr Asn Ile Asn Glu 180 185 190 Thr Ile Gly Ala Phe Gln Leu His Ile Thr Asp Lys Pro Ser Ile Asn 195 200 205 Thr Asp Lys Leu Lys Asn Ile Ser Ser Asn Tyr Cys Leu Gly Arg His 210 215 220 Leu Pro Cys Ile Ser Leu His Pro Trp Leu Ser Ser Pro Cys Ser Ser 225 230 235 240 Asp Ser Pro Pro Arg Pro Ser Ser Cys Leu Leu Ile Pro Ser Pro Glu 245 250 255 Asn Asn Ser Glu Arg Leu Leu Val Asp Thr Arg Arg Phe Leu Ile His 260 265 270 His Glu Asn Arg Thr Phe Pro Ser Thr Gln Leu Pro His Gln Ser Pro 275 280 285 Leu Gln Pro Leu Thr Ala Ala Ala Leu Ala Gly Ser Leu Gly Val Trp 290 295 300 Val Gln Asp Thr Pro Phe Ser Thr Pro Ser His Leu Phe Thr Leu His 305 310 315 320 Leu Gln Phe Cys Leu Ala Gln Gly Leu Phe Phe Leu Cys Gly Ser Ser 325 330 335 Thr Tyr Met Cys Leu Pro Ala Asn Trp Thr Gly Thr Cys Thr Leu Val 340 345 350 Phe Leu Thr Pro Lys Ile Gln Phe Ala Asn Gly Thr Glu Glu Leu Pro 355 360 365 Val Pro Leu Met Thr Pro Thr Gln Gln Lys Arg Val Ile Pro Leu Ile 370 375 380 Pro Leu Met Val Gly Leu Gly Leu Ser Ala Ser Thr Val Ala Leu Gly 385 390 395 400 Thr Gly Ile Ala Gly Ile Ser Thr Ser Val Met Thr Phe Arg Ser Leu 405 410 415 Ser Asn Asp Phe Ser Ala Ser Ile Thr Asp Ile Ser Gln Thr Leu Ser 420 425 430 Val Leu Gln Ala Gln Val Asp Ser Leu Ala Ala Val Val Leu Gln Asn 435 440 445 Arg Arg Gly Leu Asp Leu Leu Thr Ala Glu Lys Gly Gly Leu Cys Ile 450 455 460 Phe Leu Asn Glu Glu Cys Cys Phe Tyr Leu Asn Gln Ser Gly Leu Val 465 470 475 480 Tyr Asp Asn Ile Lys Lys Leu Lys Asp Arg Ala Gln Lys Leu Ala Asn 485 490 495 Gln Ala Ser Asn Tyr Ala Glu Pro Pro Trp Ala Leu Ser Asn Trp Met 500 505 510 Ser Trp Val Leu Pro Ile Val Ser Pro Leu Ile Pro Ile Phe Leu Leu 515 520 525 Leu Leu Phe Gly Pro Cys Ile Phe Arg Leu Val Ser Gln Phe Ile Gln 530 535 540 Asn Arg Ile Gln Ala Ile Thr Asn His Ser Ile Arg Gln Met Phe Leu 545 550 555 560 Leu Thr Ser Pro Gln Tyr His Pro Leu Pro Gln Asp Leu Pro Ser Ala 565 570 575 5 14 PRT homo sapiens 5 Asp Val Thr Asn Glu Ser Arg Asn Asp Asp Asp Asp Thr Ser 1 5 10 6 24 DNA Artificial Sequence FLAG sequence 6 gattacaagg atgacgacga taag 24 7 14 DNA Artificial Sequence primer 7 ttttgatcaa gctt 14 8 42 DNA Artificial Sequence adaptor 8 ctaatacgac tcactatagg gctcgagcgg ccgcccgggc ag 42 9 12 DNA Artificial Sequence adaptor 9 ggcccgtcct ag 12 10 40 DNA Artificial Sequence adaptor 10 gtaatacgac tcactatagg gcagcgtggt cgcggccgag 40 11 10 DNA Artificial Sequence adaptor 11 cggctcctag 10 12 22 DNA Artificial Sequence primer 12 ctaatacgac tcactatagg gc 22 13 22 DNA Artificial Sequence primer 13 tcgagcggcc gcccgggcag ga 22 14 20 DNA Artificial Sequence primer 14 agcgtggtcg cggccgagga 20 15 9 PRT homo sapiens 15 Asn Ala Glu Gln Pro Glu Leu Val Phe 1 5 16 9 PRT homo sapiens 16 Leu Thr Glu Ala His Gly Lys Trp Arg 1 5 17 9 PRT homo sapiens 17 Ser Met Glu Ala Gln Gly Leu Ser Phe 1 5 18 9 PRT homo sapiens 18 Asp Thr Asp Asn Pro Pro Leu Tyr Cys 1 5 19 9 PRT homo sapiens 19 Ser Thr Asp Gly Thr Phe Met Pro Ser 1 5 20 9 PRT homo sapiens 20 Ser Ile Asp Val Thr Asn Glu Ser Arg 1 5 21 9 PRT homo sapiens 21 Tyr Leu Val Pro Ser Leu Thr Arg Tyr 1 5 22 9 PRT homo sapiens 22 Ala Leu Phe Pro Ser Leu Gly Thr Tyr 1 5 23 9 PRT homo sapiens 23 Val Leu Asp Ile Pro Thr Thr Gln Arg 1 5 24 9 PRT homo sapiens 24 Leu Leu Asp Trp Gln Gly Ile Phe Ala 1 5 25 9 PRT homo sapiens 25 Cys Leu Phe Ile Phe Val Leu Ile Tyr 1 5 26 9 PRT homo sapiens 26 Thr Gln Asp Tyr Lys Trp Val Asp Arg 1 5 27 9 PRT homo sapiens 27 Val Ser Glu Thr Ser Cys Gln Val Ser 1 5 28 9 PRT homo sapiens 28 Tyr Ser Glu Glu Ile Lys Ser Asn Ile 1 5 29 9 PRT homo sapiens 29 Ile Gly Leu Pro Asn Thr Gln Asp Tyr 1 5 30 9 PRT homo sapiens 30 Trp Thr Tyr Ser Gly Gln Trp Met Tyr 1 5 31 9 PRT homo sapiens 31 Leu Ile Tyr Val Arg Val Phe Arg Lys 1 5 32 9 PRT homo sapiens 32 Gln Thr Ala Cys Gly Thr Val Gly Lys 1 5 33 9 PRT homo sapiens 33 Ser Leu Gly Thr Tyr Asp Leu Glu Lys 1 5 34 9 PRT homo sapiens 34 Thr Leu Glu Ala His Gln Ser Lys Val 1 5 35 9 PRT homo sapiens 35 Leu His Glu Ala Ser Glu Asn Leu Lys 1 5 36 9 PRT homo sapiens 36 Leu Leu Glu Gly Asn Phe Ser Leu Cys 1 5 37 9 PRT homo sapiens 37 Cys Val Glu Asn Lys Asn Gly Ser Gly 1 5 38 9 PRT homo sapiens 38 Gln Ala Glu Val Gln Asn His Ser Thr 1 5 39 9 PRT homo sapiens 39 Asn Ser Gln Pro Leu Asn Leu Ala Leu 1 5 40 9 PRT homo sapiens 40 Ala Ser Ala Ser Thr Trp Trp Thr Tyr 1 5 41 9 PRT homo sapiens 41 Trp Ser Gly Asn Asp Thr Cys Leu Tyr 1 5 42 9 PRT homo sapiens 42 Gln Asn Gln Thr Lys Gly Leu Leu Tyr 1 5 43 9 PRT homo sapiens 43 Leu Val Gln Pro Gln His Leu Leu Ala 1 5 44 9 PRT homo sapiens 44 Gln Thr Leu Glu Ala His Gln Ser Lys 1 5 45 9 PRT homo sapiens 45 Phe Leu Gly Asn Ile Pro Lys Gln Tyr 1 5 46 9 PRT homo sapiens 46 Ile Val Leu Phe Cys Leu Phe Ile Phe 1 5 47 9 PRT homo sapiens 47 Ser Val Asn Asn Ser Gly Leu Phe Phe 1 5 48 9 PRT homo sapiens 48 Asp Leu Glu Lys Ala Ile Leu Asn Ile 1 5 49 9 PRT homo sapiens 49 Ala Met Glu Gln Glu Phe Ser Ala Thr 1 5 50 9 PRT homo sapiens 50 Gln Pro Glu Leu Val Phe Val Pro Ala 1 5 51 9 PRT homo sapiens 51 Phe Leu Cys Gly Asn Gly Val Tyr Lys 1 5 52 9 PRT homo sapiens 52 Trp Ser Gly Arg Cys Gly Leu Gly Tyr 1 5 53 9 PRT homo sapiens 53 Lys Gln Cys Cys Leu Tyr Ile Asn Tyr 1 5 54 9 PRT homo sapiens 54 Ser Ser Leu Ala Ser Ala Ser Arg Lys 1 5 55 9 PRT homo sapiens 55 Val Ser Ser Leu Ala Ser Ala Ser Arg 1 5 56 9 PRT homo sapiens 56 Tyr Ser Gly Gln Trp Met Tyr Glu Arg 1 5 57 9 PRT homo sapiens 57 Gly Pro Phe Leu Gly Asn Ile Pro Lys 1 5 58 9 PRT homo sapiens 58 Gln Gly Asp Thr Asp Asn Pro Pro Leu 1 5 59 9 PRT homo sapiens 59 Asp Thr Ser Val Cys Leu Gly Thr Arg 1 5 60 9 PRT homo sapiens 60 Gly Asn Asp Thr Cys Leu Tyr Ser Cys 1 5 61 9 PRT homo sapiens 61 Glu Thr Ser Cys Gln Val Ser Asn Arg 1 5 62 9 PRT homo sapiens 62 Trp Phe Asp Ser Thr Asp Gly Thr Phe 1 5 63 9 PRT homo sapiens 63 Phe Ile Phe Val Leu Ile Tyr Val Arg 1 5 64 9 PRT homo sapiens 64 Val Leu Ile Tyr Val Arg Val Phe Arg 1 5 65 10 PRT homo sapiens 65 Leu Leu Asp Trp Gln Gly Ile Phe Ala Lys 1 5 10 66 10 PRT homo sapiens 66 Ala Met Glu Gln Glu Phe Ser Ala Thr Lys 1 5 10 67 10 PRT homo sapiens 67 Gln Gly Asp Thr Asp Asn Pro Pro Leu Tyr 1 5 10 68 10 PRT homo sapiens 68 Cys Ala Asp Ala Ser Ile Thr Asn Asp Lys 1 5 10 69 10 PRT homo sapiens 69 Val Gly Asp Trp Phe Arg Ser Trp Gly Tyr 1 5 10 70 10 PRT homo sapiens 70 Leu Leu Glu Gly Asn Phe Ser Leu Cys Val 1 5 10 71 10 PRT homo sapiens 71 Ala Ser Glu Asn Leu Lys Asn Val Pro Leu 1 5 10 72 10 PRT homo sapiens 72 Val Ser Glu Thr Ser Cys Gln Val Ser Asn 1 5 10 73 10 PRT homo sapiens 73 Ser Thr Asp Gly Thr Phe Met Pro Ser Ile 1 5 10 74 10 PRT homo sapiens 74 Asp Thr Asp Asn Pro Pro Leu Tyr Cys Asn 1 5 10 75 10 PRT homo sapiens 75 Ile Thr Asn Leu Arg Ser Phe Ile His Lys 1 5 10 76 10 PRT homo sapiens 76 Phe Cys Leu Phe Ile Phe Val Leu Ile Tyr 1 5 10 77 10 PRT homo sapiens 77 Ser Glu Glu Ile Lys Ser Asn Ile Gln Arg 1 5 10 78 10 PRT homo sapiens 78 Leu Thr Glu Ala His Gly Lys Trp Arg Cys 1 5 10 79 10 PRT homo sapiens 79 Leu Cys Glu His Leu Asp Asn Ala Glu Gln 1 5 10 80 10 PRT homo sapiens 80 Asn Ala Glu Gln Pro Glu Leu Val Phe Val 1 5 10 81 10 PRT homo sapiens 81 Tyr Ser Glu Glu Ile Lys Ser Asn Ile Gln 1 5 10 82 10 PRT homo sapiens 82 Gly Thr Val Gly Lys Gln Cys Cys Leu Tyr 1 5 10 83 10 PRT homo sapiens 83 Val Leu Ile Tyr Val Arg Val Phe Arg Lys 1 5 10 84 10 PRT homo sapiens 84 His Leu Asp Asn Ala Glu Gln Pro Glu Leu 1 5 10 85 10 PRT homo sapiens 85 Gln Ala Glu Val Gln Asn His Ser Thr Ser 1 5 10 86 10 PRT homo sapiens 86 Thr Leu Glu Ala His Gln Ser Lys Val Ser 1 5 10 87 10 PRT homo sapiens 87 Ala Ser Met Glu Ala Gln Gly Leu Ser Phe 1 5 10 88 10 PRT homo sapiens 88 Leu Ser Val Asn Asn Ser Gly Leu Phe Phe 1 5 10 89 10 PRT homo sapiens 89 Ile Gly Leu Pro Asn Thr Gln Asp Tyr Lys 1 5 10 90 10 PRT homo sapiens 90 Ser Ile Asp Val Thr Asn Glu Ser Arg Asn 1 5 10 91 10 PRT homo sapiens 91 Leu Ile Gly Leu Pro Asn Thr Gln Asp Tyr 1 5 10 92 10 PRT homo sapiens 92 Val Leu Asp Ile Pro Thr Thr Gln Arg Gln 1 5 10 93 10 PRT homo sapiens 93 Asp Asn Pro Pro Leu Tyr Cys Asn Pro Lys 1 5 10 94 10 PRT homo sapiens 94 Arg Ala Leu Phe Pro Ser Leu Gly Thr Tyr 1 5 10 95 10 PRT homo sapiens 95 Asn Thr Gln Asp Tyr Lys Trp Val Asp Arg 1 5 10 96 10 PRT homo sapiens 96 Leu Ile Val Leu Phe Cys Leu Phe Ile Phe 1 5 10 97 10 PRT homo sapiens 97 Glu Val Gln Asn His Ser Thr Ser Ser Tyr 1 5 10 98 10 PRT homo sapiens 98 Ser Ala Val Pro Leu Ile Gly Leu Pro Asn 1 5 10 99 10 PRT homo sapiens 99 Gln Pro Glu Leu Val Phe Val Pro Ala Ser 1 5 10 100 10 PRT homo sapiens 100 Ser Met Glu Ala Gln Gly Leu Ser Phe Ala 1 5 10 101 10 PRT homo sapiens 101 Ser Cys Gln Val Ser Asn Arg Ala Met Lys 1 5 10 102 10 PRT homo sapiens 102 Cys Gln Asn Gln Thr Lys Gly Leu Leu Tyr 1 5 10 103 10 PRT homo sapiens 103 Val Ser Ser Leu Ala Ser Ala Ser Arg Lys 1 5 10 104 10 PRT homo sapiens 104 Asn Ser Gln Pro Leu Asn Leu Ala Leu Ser 1 5 10 105 10 PRT homo sapiens 105 Trp Val Asp Arg Asn Ser Gly Leu Thr Trp 1 5 10 106 10 PRT homo sapiens 106 Lys Gly Phe Pro Pro Lys Trp Ser Gly Arg 1 5 10 107 10 PRT homo sapiens 107 Ser Gly Pro Phe Leu Gly Asn Ile Pro Lys 1 5 10 108 10 PRT homo sapiens 108 Leu His Glu Ala Ser Glu Asn Leu Lys Asn 1 5 10 109 10 PRT homo sapiens 109 Lys Val Ser Ser Leu Ala Ser Ala Ser Arg 1 5 10 110 10 PRT homo sapiens 110 Phe Val Leu Ile Tyr Val Arg Val Phe Arg 1 5 10 111 10 PRT homo sapiens 111 Arg Leu His Glu Ala Ser Glu Asn Leu Lys 1 5 10 112 10 PRT homo sapiens 112 Cys Leu Tyr Ser Cys Gln Asn Gln Thr Lys 1 5 10 113 10 PRT homo sapiens 113 Ala Leu Ser Pro Gln Gln Ser Ala Gln Leu 1 5 10 114 10 PRT homo sapiens 114 Pro Ser Leu Gly Thr Tyr Asp Leu Glu Lys 1 5 10 115 9 PRT homo sapiens 115 Val Leu Phe Cys Leu Phe Ile Phe Val 1 5 116 9 PRT homo sapiens 116 Arg Leu Leu Glu Gly Asn Phe Ser Leu 1 5 117 9 PRT homo sapiens 117 Val Leu Leu Ile Val Leu Phe Cys Leu 1 5 118 9 PRT homo sapiens 118 Trp Leu Cys Glu His Leu Asp Asn Ala 1 5 119 9 PRT homo sapiens 119 Trp Leu Thr Gly Ser Asn Leu Thr Leu 1 5 120 9 PRT homo sapiens 120 Tyr Val Leu Leu Ile Val Leu Phe Cys 1 5 121 9 PRT homo sapiens 121 Thr Leu Thr Ala Phe Leu Thr Ile Leu 1 5 122 9 PRT homo sapiens 122 Leu Gln Leu Thr Leu Thr Ala Phe Leu 1 5 123 9 PRT homo sapiens 123 Trp Met Tyr Glu Arg Val Trp Tyr Pro 1 5 124 9 PRT homo sapiens 124 Leu Ile Val Leu Phe Cys Leu Phe Ile 1 5 125 9 PRT homo sapiens 125 Arg Val Trp Tyr Pro Gln Ala Glu Val 1 5 126 9 PRT homo sapiens 126 Gly Leu Gly Tyr Leu Val Pro Ser Leu 1 5 127 9 PRT homo sapiens 127 Tyr Leu Thr Leu Asn Ala Ser Gln Ile 1 5 128 9 PRT homo sapiens 128 Tyr Gln Leu Phe Arg Asn Leu Phe Cys 1 5 129 9 PRT homo sapiens 129 Asn Leu Arg Ser Phe Ile His Lys Val 1 5 130 9 PRT homo sapiens 130 Leu Leu Tyr Gln Leu Phe Arg Asn Leu 1 5 131 9 PRT homo sapiens 131 Gln Leu Thr Leu Thr Ala Phe Leu Thr 1 5 132 9 PRT homo sapiens 132 Arg Leu His Glu Ala Ser Glu Asn Leu 1 5 133 9 PRT homo sapiens 133 Leu Val Ser Glu Thr Ser Cys Gln Val 1 5 134 9 PRT homo sapiens 134 Ile Leu Val Gln Pro Gln His Leu Leu 1 5 135 9 PRT homo sapiens 135 Cys Leu Tyr Ser Cys Gln Asn Gln Thr 1 5 136 9 PRT homo sapiens 136 Gly Leu Ser Phe Ala Gln Val Arg Leu 1 5 137 9 PRT homo sapiens 137 Ser Leu Asn Ser Gln Pro Leu Asn Leu 1 5 138 9 PRT homo sapiens 138 Thr Leu Ser Val Asn Asn Ser Gly Leu 1 5 139 9 PRT homo sapiens 139 Lys Ala Met Glu Gln Glu Phe Ser Ala 1 5 140 9 PRT homo sapiens 140 Leu Leu Asp Trp Gln Gly Ile Phe Ala 1 5 141 9 PRT homo sapiens 141 Gln Leu Leu Val Ser Glu Thr Ser Cys 1 5 142 9 PRT homo sapiens 142 Ala Leu Leu Gln Leu Thr Leu Thr Ala 1 5 143 9 PRT homo sapiens 143 Val Asn Asn Ser Gly Leu Phe Phe Leu 1 5 144 9 PRT homo sapiens 144 Ala Glu Gln Pro Glu Leu Val Phe Val 1 5 145 9 PRT homo sapiens 145 Leu Leu Ala Pro Val Phe Arg Thr Leu 1 5 146 9 PRT homo sapiens 146 Ala Gln Gly Leu Ser Phe Ala Gln Val 1 5 147 9 PRT homo sapiens 147 His Leu Leu Ala Pro Val Phe Arg Thr 1 5 148 9 PRT homo sapiens 148 Thr Ile Leu Val Gln Pro Gln His Leu 1 5 149 9 PRT homo sapiens 149 Leu Glu Gly Asn Phe Ser Leu Cys Val 1 5 150 9 PRT homo sapiens 150 Gln Ile Thr Asn Leu Arg Ser Phe Ile 1 5 151 9 PRT homo sapiens 151 Gly Leu Thr Trp Ser Gly Asn Asp Thr 1 5 152 9 PRT homo sapiens 152 Gly Leu Thr Thr His Gln Tyr Asp Thr 1 5 153 9 PRT homo sapiens 153 Asn Leu Phe Cys Ser Tyr Gly Leu Thr 1 5 154 9 PRT homo sapiens 154 Thr Leu Glu Ala His Gln Ser Lys Val 1 5 155 9 PRT homo sapiens 155 Gln Leu Phe Arg Asn Leu Phe Cys Ser 1 5 156 9 PRT homo sapiens 156 Gly Leu Leu Tyr Gln Leu Phe Arg Asn 1 5 157 9 PRT homo sapiens 157 Gly Thr Phe Met Pro Ser Ile Asp Val 1 5 158 9 PRT homo sapiens 158 Leu Val Pro Ser Leu Thr Arg Tyr Leu 1 5 159 9 PRT homo sapiens 159 Leu Thr Asn Gln Ser Asn Cys Trp Leu 1 5 160 9 PRT homo sapiens 160 Ser Ile Leu Thr Asn Gln Ser Asn Cys 1 5 161 9 PRT homo sapiens 161 Ala Ile Leu Asn Ile Ser Lys Ala Met 1 5 162 9 PRT homo sapiens 162 Phe Cys Leu Phe Ile Phe Val Leu Ile 1 5 163 9 PRT homo sapiens 163 Met Glu Ala Gln Gly Leu Ser Phe Ala 1 5 164 9 PRT homo sapiens 164 Trp Gly Tyr Val Leu Leu Ile Val Leu 1 5 165 10 PRT homo sapiens 165 Cys Leu Phe Ile Phe Val Leu Ile Tyr Val 1 5 10 166 10 PRT homo sapiens 166 Val Leu Phe Cys Leu Phe Ile Phe Val Leu 1 5 10 167 10 PRT homo sapiens 167 Tyr Val Leu Leu Ile Val Leu Phe Cys Leu 1 5 10 168 10 PRT homo sapiens 168 Ile Val Leu Phe Cys Leu Phe Ile Phe Val 1 5 10 169 10 PRT homo sapiens 169 Leu Leu Ile Val Leu Phe Cys Leu Phe Ile 1 5 10 170 10 PRT homo sapiens 170 Gly Leu Phe Phe Leu Cys Gly Asn Gly Val 1 5 10 171 10 PRT homo sapiens 171 Phe Ile Phe Val Leu Ile Tyr Val Arg Val 1 5 10 172 10 PRT homo sapiens 172 Ile Leu Thr Asn Gln Ser Asn Cys Trp Leu 1 5 10 173 10 PRT homo sapiens 173 Leu Leu Gln Leu Thr Leu Thr Ala Phe Leu 1 5 10 174 10 PRT homo sapiens 174 Arg Leu Leu Glu Gly Asn Phe Ser Leu Cys 1 5 10 175 10 PRT homo sapiens 175 Ser Val Asn Asn Ser Gly Leu Phe Phe Leu 1 5 10 176 10 PRT homo sapiens 176 Phe Leu Gly Asn Ile Pro Lys Gln Tyr Cys 1 5 10 177 10 PRT homo sapiens 177 Leu Leu Val Ser Glu Thr Ser Cys Gln Val 1 5 10 178 10 PRT homo sapiens 178 Tyr Leu Val Pro Ser Leu Thr Arg Tyr Leu 1 5 10 179 10 PRT homo sapiens 179 Thr Leu Thr Ala Phe Leu Thr Ile Leu Val 1 5 10 180 10 PRT homo sapiens 180 Ile Leu Trp Phe Asp Ser Thr Asp Gly Thr 1 5 10 181 10 PRT homo sapiens 181 Thr Leu Asn Ala Ser Gln Ile Thr Asn Leu 1 5 10 182 10 PRT homo sapiens 182 Ser Leu Ser Asn Cys Ala Leu Leu Gln Leu 1 5 10 183 10 PRT homo sapiens 183 Gly Leu Leu Tyr Gln Leu Phe Arg Asn Leu 1 5 10 184 10 PRT homo sapiens 184 Leu Leu Glu Gly Asn Phe Ser Leu Cys Val 1 5 10 185 10 PRT homo sapiens 185 Val Gln Pro Gln His Leu Leu Ala Pro Val 1 5 10 186 10 PRT homo sapiens 186 Leu Gln Leu Thr Leu Thr Ala Phe Leu Thr 1 5 10 187 10 PRT homo sapiens 187 Ala Leu Ser Pro Gln Gln Ser Ala Gln Leu 1 5 10 188 10 PRT homo sapiens 188 Gln Leu Thr Leu Thr Ala Phe Leu Thr Ile 1 5 10 189 10 PRT homo sapiens 189 Cys Leu Tyr Ile Asn Tyr Ser Glu Glu Ile 1 5 10 190 10 PRT homo sapiens 190 Thr Asn Leu Arg Ser Phe Ile His Lys Val 1 5 10 191 10 PRT homo sapiens 191 Gln Thr Leu Glu Ala His Gln Ser Lys Val 1 5 10 192 10 PRT homo sapiens 192 Tyr Leu Thr Leu Asn Ala Ser Gln Ile Thr 1 5 10 193 10 PRT homo sapiens 193 Ser Leu Asn Ser Gln Pro Leu Asn Leu Ala 1 5 10 194 10 PRT homo sapiens 194 Thr Ile Leu Val Gln Pro Gln His Leu Leu 1 5 10 195 10 PRT homo sapiens 195 Lys Ala Met Glu Gln Glu Phe Ser Ala Thr 1 5 10 196 10 PRT homo sapiens 196 Gly Leu Ser Phe Ala Gln Val Arg Leu Leu 1 5 10 197 10 PRT homo sapiens 197 Pro Leu Leu Asp Trp Gln Gly Ile Phe Ala 1 5 10 198 10 PRT homo sapiens 198 Thr Gln Gly Asp Thr Asp Asn Pro Pro Leu 1 5 10 199 10 PRT homo sapiens 199 Ile Leu Val Gln Pro Gln His Leu Leu Ala 1 5 10 200 10 PRT homo sapiens 200 Gly Leu Gly Tyr Leu Val Pro Ser Leu Thr 1 5 10 201 10 PRT homo sapiens 201 Leu Thr Leu Thr Ala Phe Leu Thr Ile Leu 1 5 10 202 10 PRT homo sapiens 202 Leu Pro Asn Thr Gln Asp Tyr Lys Trp Val 1 5 10 203 10 PRT homo sapiens 203 Ser Gln Ile Thr Asn Leu Arg Ser Phe Ile 1 5 10 204 10 PRT homo sapiens 204 Ser Leu Gly Thr Tyr Asp Leu Glu Lys Ala 1 5 10 205 10 PRT homo sapiens 205 Thr Val Gly Lys Gln Cys Cys Leu Tyr Ile 1 5 10 206 10 PRT homo sapiens 206 Asn Gln Thr Lys Gly Leu Leu Tyr Gln Leu 1 5 10 207 10 PRT homo sapiens 207 Ser Leu Thr Arg Tyr Leu Thr Leu Asn Ala 1 5 10 208 10 PRT homo sapiens 208 Asn Leu Ala Leu Ser Pro Gln Gln Ser Ala 1 5 10 209 10 PRT homo sapiens 209 Gln Val Ser Asn Arg Ala Met Lys Gly Leu 1 5 10 210 10 PRT homo sapiens 210 Leu Thr Trp Ser Gly Asn Asp Thr Cys Leu 1 5 10 211 10 PRT homo sapiens 211 Leu Val Phe Val Pro Ala Ser Ala Ser Thr 1 5 10 212 10 PRT homo sapiens 212 Arg Ser Trp Gly Tyr Val Leu Leu Ile Val 1 5 10 213 10 PRT homo sapiens 213 Val Pro Ala Ser Ala Ser Thr Trp Trp Thr 1 5 10 214 10 PRT homo sapiens 214 Phe Ile His Lys Val Thr Pro His Arg Cys 1 5 10 215 9 PRT homo sapiens 215 Cys Leu Phe Ile Phe Val Leu Ile Tyr 1 5 216 9 PRT homo sapiens 216 Ser Leu Gly Thr Tyr Asp Leu Glu Lys 1 5 217 9 PRT homo sapiens 217 Gly Val Tyr Lys Gly Phe Pro Pro Lys 1 5 218 9 PRT homo sapiens 218 Leu Ile Tyr Val Arg Val Phe Arg Lys 1 5 219 9 PRT homo sapiens 219 Gly Leu Pro Asn Thr Gln Asp Tyr Lys 1 5 220 9 PRT homo sapiens 220 Phe Leu Cys Gly Asn Gly Val Tyr Lys 1 5 221 9 PRT homo sapiens 221 Tyr Leu Val Pro Ser Leu Thr Arg Tyr 1 5 222 9 PRT homo sapiens 222 Ala Leu Phe Pro Ser Leu Gly Thr Tyr 1 5 223 9 PRT homo sapiens 223 Val Leu Ile Tyr Val Arg Val Phe Arg 1 5 224 9 PRT homo sapiens 224 Leu Leu Ile Val Leu Phe Cys Leu Phe 1 5 225 9 PRT homo sapiens 225 Phe Ile Phe Val Leu Ile Tyr Val Arg 1 5 226 9 PRT homo sapiens 226 Val Leu Leu Ile Val Leu Phe Cys Leu 1 5 227 9 PRT homo sapiens 227 Val Leu Asp Ile Pro Thr Thr Gln Arg 1 5 228 9 PRT homo sapiens 228 Gly Pro Phe Leu Gly Asn Ile Pro Lys 1 5 229 9 PRT homo sapiens 229 Val Leu Phe Cys Leu Phe Ile Phe Val 1 5 230 9 PRT homo sapiens 230 Gly Leu Gly Tyr Leu Val Pro Ser Leu 1 5 231 9 PRT homo sapiens 231 Arg Leu Leu Glu Gly Asn Phe Ser Leu 1 5 232 9 PRT homo sapiens 232 Cys Leu Gly Thr Arg Gln Cys Ser Arg 1 5 233 9 PRT homo sapiens 233 Ser Met Glu Ala Gln Gly Leu Ser Phe 1 5 234 9 PRT homo sapiens 234 Phe Leu Gly Asn Ile Pro Lys Gln Tyr 1 5 235 9 PRT homo sapiens 235 Trp Thr Tyr Ser Gly Gln Trp Met Tyr 1 5 236 9 PRT homo sapiens 236 Leu Leu Gln Leu Thr Leu Thr Ala Phe 1 5 237 9 PRT homo sapiens 237 Thr Leu Thr Ala Phe Leu Thr Ile Leu 1 5 238 9 PRT homo sapiens 238 Gln Thr Leu Glu Ala His Gln Ser Lys 1 5 239 9 PRT homo sapiens 239 His Leu Leu Ala Pro Val Phe Arg Thr 1 5 240 9 PRT homo sapiens 240 Tyr Ile Asn Tyr Ser Glu Glu Ile Lys 1 5 241 9 PRT homo sapiens 241 Gly Leu Ser Phe Ala Gln Val Arg Leu 1 5 242 9 PRT homo sapiens 242 Trp Leu Thr Gly Ser Asn Leu Thr Leu 1 5 243 9 PRT homo sapiens 243 Ile Leu Val Gln Pro Gln His Leu Leu 1 5 244 9 PRT homo sapiens 244 Trp Met Tyr Glu Arg Val Trp Tyr Pro 1 5 245 9 PRT homo sapiens 245 Ile Val Leu Phe Cys Leu Phe Ile Phe 1 5 246 9 PRT homo sapiens 246 Ser Leu Asn Ser Gln Pro Leu Asn Leu 1 5 247 9 PRT homo sapiens 247 Gln Thr Ala Cys Gly Thr Val Gly Lys 1 5 248 9 PRT homo sapiens 248 Gly Leu Thr Thr His Gln Tyr Asp Thr 1 5 249 9 PRT homo sapiens 249 Leu Leu Glu Gly Asn Phe Ser Leu Cys 1 5 250 9 PRT homo sapiens 250 Asn Leu Arg Ser Phe Ile His Lys Val 1 5 251 9 PRT homo sapiens 251 Leu Leu Tyr Gln Leu Phe Arg Asn Leu 1 5 252 9 PRT homo sapiens 252 Arg Leu His Glu Ala Ser Glu Asn Leu 1 5 253 9 PRT homo sapiens 253 Cys Gln Val Ser Asn Arg Ala Met Lys 1 5 254 9 PRT homo sapiens 254 Gln Leu Phe Arg Asn Leu Phe Cys Ser 1 5 255 9 PRT homo sapiens 255 Lys Gln Cys Cys Leu Tyr Ile Asn Tyr 1 5 256 9 PRT homo sapiens 256 Thr Leu Ser Val Asn Asn Ser Gly Leu 1 5 257 9 PRT homo sapiens 257 Asn Pro Pro Leu Tyr Cys Asn Pro Lys 1 5 258 9 PRT homo sapiens 258 Phe Ile His Lys Val Thr Pro His Arg 1 5 259 9 PRT homo sapiens 259 Tyr Leu Thr Leu Asn Ala Ser Gln Ile 1 5 260 9 PRT homo sapiens 260 Tyr Val Arg Val Phe Arg Lys Ser Arg 1 5 261 9 PRT homo sapiens 261 Ala Leu Leu Gln Leu Thr Leu Thr Ala 1 5 262 9 PRT homo sapiens 262 Leu Ile Val Leu Phe Cys Leu Phe Ile 1 5 263 9 PRT homo sapiens 263 Asp Leu Glu Lys Ala Ile Leu Asn Ile 1 5 264 9 PRT homo sapiens 264 Cys Leu Tyr Ser Cys Gln Asn Gln Thr 1 5 265 10 PRT homo sapiens 265 Val Leu Ile Tyr Val Arg Val Phe Arg Lys 1 5 10 266 10 PRT homo sapiens 266 Cys Leu Tyr Ser Cys Gln Asn Gln Thr Lys 1 5 10 267 10 PRT homo sapiens 267 Leu Leu Asp Trp Gln Gly Ile Phe Ala Lys 1 5 10 268 10 PRT homo sapiens 268 Ala Met Glu Gln Glu Phe Ser Ala Thr Lys 1 5 10 269 10 PRT homo sapiens 269 Gln Leu Phe Arg Asn Leu Phe Cys Ser Tyr 1 5 10 270 10 PRT homo sapiens 270 Val Leu Phe Cys Leu Phe Ile Phe Val Leu 1 5 10 271 10 PRT homo sapiens 271 Arg Leu His Glu Ala Ser Glu Asn Leu Lys 1 5 10 272 10 PRT homo sapiens 272 Leu Leu Tyr Gln Leu Phe Arg Asn Leu Phe 1 5 10 273 10 PRT homo sapiens 273 Gly Leu Thr Glu Ala His Gly Lys Trp Arg 1 5 10 274 10 PRT homo sapiens 274 Val Leu Leu Ile Val Leu Phe Cys Leu Phe 1 5 10 275 10 PRT homo sapiens 275 Cys Leu Tyr Ile Asn Tyr Ser Glu Glu Ile 1 5 10 276 10 PRT homo sapiens 276 Ile Thr Asn Leu Arg Ser Phe Ile His Lys 1 5 10 277 10 PRT homo sapiens 277 Ala Met Lys Gly Leu Thr Thr His Gln Tyr 1 5 10 278 10 PRT homo sapiens 278 Leu Leu Ile Val Leu Phe Cys Leu Phe Ile 1 5 10 279 10 PRT homo sapiens 279 Cys Leu Phe Ile Phe Val Leu Ile Tyr Val 1 5 10 280 10 PRT homo sapiens 280 Ala Leu Leu Gln Leu Thr Leu Thr Ala Phe 1 5 10 281 10 PRT homo sapiens 281 Gly Ile Phe Ala Lys Val Gly Asp Trp Phe 1 5 10 282 10 PRT homo sapiens 282 Gln Leu Thr Leu Thr Ala Phe Leu Thr Ile 1 5 10 283 10 PRT homo sapiens 283 Gly Leu Phe Phe Leu Cys Gly Asn Gly Val 1 5 10 284 10 PRT homo sapiens 284 Lys Gln Tyr Cys Asn Gln Ile Leu Trp Phe 1 5 10 285 10 PRT homo sapiens 285 Cys Leu Gly Thr Arg Gln Cys Ser Arg Phe 1 5 10 286 10 PRT homo sapiens 286 Gly Asn Phe Ser Leu Cys Val Glu Asn Lys 1 5 10 287 10 PRT homo sapiens 287 Gly Gln Trp Met Tyr Glu Arg Val Trp Tyr 1 5 10 288 10 PRT homo sapiens 288 Leu Leu Glu Gly Asn Phe Ser Leu Cys Val 1 5 10 289 10 PRT homo sapiens 289 Ser Leu Ser Asn Cys Ala Leu Leu Gln Leu 1 5 10 290 10 PRT homo sapiens 290 Lys Gln Thr Leu Glu Ala His Gln Ser Lys 1 5 10 291 10 PRT homo sapiens 291 Gly Leu Pro Asn Thr Gln Asp Tyr Lys Trp 1 5 10 292 10 PRT homo sapiens 292 Leu Ile Val Leu Phe Cys Leu Phe Ile Phe 1 5 10 293 10 PRT homo sapiens 293 Arg Leu Leu Glu Gly Asn Phe Ser Leu Cys 1 5 10 294 10 PRT homo sapiens 294 Gly Thr Val Gly Lys Gln Cys Cys Leu Tyr 1 5 10 295 10 PRT homo sapiens 295 Lys Val Ser Ser Leu Ala Ser Ala Ser Arg 1 5 10 296 10 PRT homo sapiens 296 Phe Cys Leu Phe Ile Phe Val Leu Ile Tyr 1 5 10 297 10 PRT homo sapiens 297 Thr Leu Asn Ala Ser Gln Ile Thr Asn Leu 1 5 10 298 10 PRT homo sapiens 298 Gly Val Tyr Lys Gly Phe Pro Pro Lys Trp 1 5 10 299 10 PRT homo sapiens 299 Ala Leu Ser Pro Gln Gln Ser Ala Gln Leu 1 5 10 300 10 PRT homo sapiens 300 Phe Val Leu Ile Tyr Val Arg Val Phe Arg 1 5 10 301 10 PRT homo sapiens 301 Asn Leu Lys Asn Val Pro Leu Leu Asp Trp 1 5 10 302 10 PRT homo sapiens 302 His Val Leu Asp Ile Pro Thr Thr Gln Arg 1 5 10 303 10 PRT homo sapiens 303 Gly Leu Leu Tyr Gln Leu Phe Arg Asn Leu 1 5 10 304 10 PRT homo sapiens 304 Trp Met Tyr Glu Arg Val Trp Tyr Pro Gln 1 5 10 305 10 PRT homo sapiens 305 Tyr Val Leu Leu Ile Val Leu Phe Cys Leu 1 5 10 306 10 PRT homo sapiens 306 His Leu Asp Asn Ala Glu Gln Pro Glu Leu 1 5 10 307 10 PRT homo sapiens 307 Gly Leu Thr Trp Ser Gly Asn Asp Thr Cys 1 5 10 308 10 PRT homo sapiens 308 Asn Leu Phe Cys Ser Tyr Gly Leu Thr Glu 1 5 10 309 10 PRT homo sapiens 309 Ile Leu Val Gln Pro Gln His Leu Leu Ala 1 5 10 310 10 PRT homo sapiens 310 Leu Leu Gln Leu Thr Leu Thr Ala Phe Leu 1 5 10 311 10 PRT homo sapiens 311 Thr Leu Ser Val Asn Asn Ser Gly Leu Phe 1 5 10 312 10 PRT homo sapiens 312 Ile Leu Thr Asn Gln Ser Asn Cys Trp Leu 1 5 10 313 10 PRT homo sapiens 313 Arg Gln Thr Ala Cys Gly Thr Val Gly Lys 1 5 10 314 10 PRT homo sapiens 314 Gly Leu Ser Phe Ala Gln Val Arg Leu Leu 1 5 10 315 9 PRT homo sapiens 315 Gly Val Tyr Lys Gly Phe Pro Pro Lys 1 5 316 9 PRT homo sapiens 316 Gly Pro Phe Leu Gly Asn Ile Pro Lys 1 5 317 9 PRT homo sapiens 317 Leu Ile Tyr Val Arg Val Phe Arg Lys 1 5 318 9 PRT homo sapiens 318 Gln Thr Leu Glu Ala His Gln Ser Lys 1 5 319 9 PRT homo sapiens 319 Gly Leu Pro Asn Thr Gln Asp Tyr Lys 1 5 320 9 PRT homo sapiens 320 Gln Thr Ala Cys Gly Thr Val Gly Lys 1 5 321 9 PRT homo sapiens 321 Cys Gln Val Ser Asn Arg Ala Met Lys 1 5 322 9 PRT homo sapiens 322 Ser Leu Gly Thr Tyr Asp Leu Glu Lys 1 5 323 9 PRT homo sapiens 323 Gly Tyr Leu Val Pro Ser Leu Thr Arg 1 5 324 9 PRT homo sapiens 324 Leu Tyr Ser Cys Gln Asn Gln Thr Lys 1 5 325 9 PRT homo sapiens 325 Tyr Ile Asn Tyr Ser Glu Glu Ile Lys 1 5 326 9 PRT homo sapiens 326 Phe Leu Cys Gly Asn Gly Val Tyr Lys 1 5 327 9 PRT homo sapiens 327 Phe Ile Phe Val Leu Ile Tyr Val Arg 1 5 328 9 PRT homo sapiens 328 Asn Phe Ser Leu Cys Val Glu Asn Lys 1 5 329 9 PRT homo sapiens 329 Tyr Val Arg Val Phe Arg Lys Ser Arg 1 5 330 9 PRT homo sapiens 330 Asn Pro Pro Leu Tyr Cys Asn Pro Lys 1 5 331 9 PRT homo sapiens 331 Thr Asn Leu Arg Ser Phe Ile His Lys 1 5 332 9 PRT homo sapiens 332 Leu Asp Trp Gln Gly Ile Phe Ala Lys 1 5 333 9 PRT homo sapiens 333 Gly Thr Phe Met Pro Ser Ile Asp Val 1 5 334 9 PRT homo sapiens 334 Val Leu Ile Tyr Val Arg Val Phe Arg 1 5 335 9 PRT homo sapiens 335 Thr Gln Asp Tyr Lys Trp Val Asp Arg 1 5 336 9 PRT homo sapiens 336 Gly Phe Pro Pro Lys Trp Ser Gly Arg 1 5 337 9 PRT homo sapiens 337 Arg Val Trp Tyr Pro Gln Ala Glu Val 1 5 338 9 PRT homo sapiens 338 Leu Thr Glu Ala His Gly Lys Trp Arg 1 5 339 9 PRT homo sapiens 339 Ile Val Leu Phe Cys Leu Phe Ile Phe 1 5 340 9 PRT homo sapiens 340 Cys Leu Gly Thr Arg Gln Cys Ser Arg 1 5 341 9 PRT homo sapiens 341 Val Leu Asp Ile Pro Thr Thr Gln Arg 1 5 342 9 PRT homo sapiens 342 Phe Ile His Lys Val Thr Pro His Arg 1 5 343 9 PRT homo sapiens 343 Ser Ile Asp Val Thr Asn Glu Ser Arg 1 5 344 9 PRT homo sapiens 344 Phe Ala Lys Val Gly Asp Trp Phe Arg 1 5 345 9 PRT homo sapiens 345 Lys Gln Tyr Cys Asn Gln Ile Leu Trp 1 5 346 9 PRT homo sapiens 346 Glu Thr Ser Cys Gln Val Ser Asn Arg 1 5 347 9 PRT homo sapiens 347 Met Glu Gln Glu Phe Ser Ala Thr Lys 1 5 348 9 PRT homo sapiens 348 Asp Thr Ser Val Cys Leu Gly Thr Arg 1 5 349 9 PRT homo sapiens 349 Arg Leu Leu Glu Gly Asn Phe Ser Leu 1 5 350 9 PRT homo sapiens 350 Gly Thr Val Gly Lys Gln Cys Cys Leu 1 5 351 9 PRT homo sapiens 351 Asn Pro Lys Asp Asn Ser Thr Ile Arg 1 5 352 9 PRT homo sapiens 352 Leu Val Gln Pro Gln His Leu Leu Ala 1 5 353 9 PRT homo sapiens 353 Trp Thr Tyr Ser Gly Gln Trp Met Tyr 1 5 354 9 PRT homo sapiens 354 Ser Val Asn Asn Ser Gly Leu Phe Phe 1 5 355 9 PRT homo sapiens 355 Asn His Ser Thr Ser Ser Tyr Arg Lys 1 5 356 9 PRT homo sapiens 356 Asn Ala Ser Gln Ile Thr Asn Leu Arg 1 5 357 9 PRT homo sapiens 357 Lys Gly Leu Leu Tyr Gln Leu Phe Arg 1 5 358 9 PRT homo sapiens 358 Lys Ala Met Glu Gln Glu Phe Ser Ala 1 5 359 9 PRT homo sapiens 359 Lys Gln Cys Cys Leu Tyr Ile Asn Tyr 1 5 360 9 PRT homo sapiens 360 Tyr Gly Leu Thr Glu Ala His Gly Lys 1 5 361 9 PRT homo sapiens 361 Ser Ser Leu Ala Ser Ala Ser Arg Lys 1 5 362 9 PRT homo sapiens 362 Leu Thr Leu Thr Ala Phe Leu Thr Ile 1 5 363 9 PRT homo sapiens 363 Ile Thr Asn Leu Arg Ser Phe Ile His 1 5 364 9 PRT homo sapiens 364 Gly Thr Tyr Asp Leu Glu Lys Ala Ile 1 5 365 10 PRT homo sapiens 365 Ile Thr Asn Leu Arg Ser Phe Ile His Lys 1 5 10 366 10 PRT homo sapiens 366 Val Leu Ile Tyr Val Arg Val Phe Arg Lys 1 5 10 367 10 PRT homo sapiens 367 Arg Gln Thr Ala Cys Gly Thr Val Gly Lys 1 5 10 368 10 PRT homo sapiens 368 Lys Gln Thr Leu Glu Ala His Gln Ser Lys 1 5 10 369 10 PRT homo sapiens 369 Leu Leu Asp Trp Gln Gly Ile Phe Ala Lys 1 5 10 370 10 PRT homo sapiens 370 Lys Val Ser Ser Leu Ala Ser Ala Ser Arg 1 5 10 371 10 PRT homo sapiens 371 Arg Leu His Glu Ala Ser Glu Asn Leu Lys 1 5 10 372 10 PRT homo sapiens 372 Cys Leu Tyr Ser Cys Gln Asn Gln Thr Lys 1 5 10 373 10 PRT homo sapiens 373 Phe Val Leu Ile Tyr Val Arg Val Phe Arg 1 5 10 374 10 PRT homo sapiens 374 Leu Tyr Ile Asn Tyr Ser Glu Glu Ile Lys 1 5 10 375 10 PRT homo sapiens 375 His Val Leu Asp Ile Pro Thr Thr Gln Arg 1 5 10 376 10 PRT homo sapiens 376 Tyr Val Arg Val Phe Arg Lys Ser Arg Arg 1 5 10 377 10 PRT homo sapiens 377 Ser Tyr Gly Leu Thr Glu Ala His Gly Lys 1 5 10 378 10 PRT homo sapiens 378 Ala Met Glu Gln Glu Phe Ser Ala Thr Lys 1 5 10 379 10 PRT homo sapiens 379 Phe Phe Leu Cys Gly Asn Gly Val Tyr Lys 1 5 10 380 10 PRT homo sapiens 380 Gly Asn Phe Ser Leu Cys Val Glu Asn Lys 1 5 10 381 10 PRT homo sapiens 381 Ser Cys Gln Val Ser Asn Arg Ala Met Lys 1 5 10 382 10 PRT homo sapiens 382 Cys Ala Asp Ala Ser Ile Thr Asn Asp Lys 1 5 10 383 10 PRT homo sapiens 383 Asn Thr Gln Asp Tyr Lys Trp Val Asp Arg 1 5 10 384 10 PRT homo sapiens 384 Thr Tyr Ser Gly Gln Trp Met Tyr Glu Arg 1 5 10 385 10 PRT homo sapiens 385 Val Gln Asn His Ser Thr Ser Ser Tyr Arg 1 5 10 386 10 PRT homo sapiens 386 Gly Val Tyr Lys Gly Phe Pro Pro Lys Trp 1 5 10 387 10 PRT homo sapiens 387 Ala Gln Gly Leu Ser Phe Ala Gln Val Arg 1 5 10 388 10 PRT homo sapiens 388 Leu Glu Lys Ala Ile Leu Asn Ile Ser Lys 1 5 10 389 10 PRT homo sapiens 389 Gly Leu Thr Glu Ala His Gly Lys Trp Arg 1 5 10 390 10 PRT homo sapiens 390 Leu Phe Ile Phe Val Leu Ile Tyr Val Arg 1 5 10 391 10 PRT homo sapiens 391 Tyr Val Leu Leu Ile Val Leu Phe Cys Leu 1 5 10 392 10 PRT homo sapiens 392 Ile Phe Ala Lys Val Gly Asp Trp Phe Arg 1 5 10 393 10 PRT homo sapiens 393 Gln Asn His Ser Thr Ser Ser Tyr Arg Lys 1 5 10 394 10 PRT homo sapiens 394 Lys Gln Tyr Cys Asn Gln Ile Leu Trp Phe 1 5 10 395 10 PRT homo sapiens 395 Arg Thr Trp Asn Ser Ser Ala Val Pro Leu 1 5 10 396 10 PRT homo sapiens 396 Ser Val Asn Asn Ser Gly Leu Phe Phe Leu 1 5 10 397 10 PRT homo sapiens 397 Lys Val Thr Trp His Trp Glu Ala Ser Met 1 5 10 398 10 PRT homo sapiens 398 Gly Thr Tyr Asp Leu Glu Lys Ala Ile Leu 1 5 10 399 10 PRT homo sapiens 399 Val Cys Leu Gly Thr Arg Gln Cys Ser Arg 1 5 10 400 10 PRT homo sapiens 400 Ile Val Leu Phe Cys Leu Phe Ile Phe Val 1 5 10 401 10 PRT homo sapiens 401 Ile Tyr Val Arg Val Phe Arg Lys Ser Arg 1 5 10 402 10 PRT homo sapiens 402 Ser Phe Ile His Lys Val Thr Pro His Arg 1 5 10 403 10 PRT homo sapiens 403 Gly Thr Val Gly Lys Gln Cys Cys Leu Tyr 1 5 10 404 10 PRT homo sapiens 404 Thr Val Gly Lys Gln Cys Cys Leu Tyr Ile 1 5 10 405 10 PRT homo sapiens 405 Trp Val Asp Arg Asn Ser Gly Leu Thr Trp 1 5 10 406 10 PRT homo sapiens 406 Ser Gly Pro Phe Leu Gly Asn Ile Pro Lys 1 5 10 407 10 PRT homo sapiens 407 Gly Gln Trp Met Tyr Glu Arg Val Trp Tyr 1 5 10 408 10 PRT homo sapiens 408 Asn Gly Val Tyr Lys Gly Phe Pro Pro Lys 1 5 10 409 10 PRT homo sapiens 409 Ile Gly Leu Pro Asn Thr Gln Asp Tyr Lys 1 5 10 410 10 PRT homo sapiens 410 Lys Gly Phe Pro Pro Lys Trp Ser Gly Arg 1 5 10 411 10 PRT homo sapiens 411 Gly Ile Phe Ala Lys Val Gly Asp Trp Phe 1 5 10 412 10 PRT homo sapiens 412 Ser Glu Glu Ile Lys Ser Asn Ile Gln Arg 1 5 10 413 10 PRT homo sapiens 413 Gly Leu Pro Asn Thr Gln Asp Tyr Lys Trp 1 5 10 414 10 PRT homo sapiens 414 Gly Leu Phe Phe Leu Cys Gly Asn Gly Val 1 5 10 415 9 PRT homo sapiens 415 Thr Tyr Asp Leu Glu Lys Ala Ile Leu 1 5 416 9 PRT homo sapiens 416 Gly Tyr Val Leu Leu Ile Val Leu Phe 1 5 417 9 PRT homo sapiens 417 Leu Tyr Gln Leu Phe Arg Asn Leu Phe 1 5 418 9 PRT homo sapiens 418 Gln Tyr Cys Asn Gln Ile Leu Trp Phe 1 5 419 9 PRT homo sapiens 419 Leu Tyr Ile Asn Tyr Ser Glu Glu Ile 1 5 420 9 PRT homo sapiens 420 Glu Phe Ser Ala Thr Lys Gln Thr Leu 1 5 421 9 PRT homo sapiens 421 Trp Phe Arg Ser Trp Gly Tyr Val Leu 1 5 422 9 PRT homo sapiens 422 Leu Phe Cys Leu Phe Ile Phe Val Leu 1 5 423 9 PRT homo sapiens 423 Val Phe Arg Lys Ser Arg Arg Ser Leu 1 5 424 9 PRT homo sapiens 424 Lys Trp Val Asp Arg Asn Ser Gly Leu 1 5 425 9 PRT homo sapiens 425 Arg Leu Leu Glu Gly Asn Phe Ser Leu 1 5 426 9 PRT homo sapiens 426 Ile Phe Ala Lys Val Gly Asp Trp Phe 1 5 427 9 PRT homo sapiens 427 Ile Tyr Val Arg Val Phe Arg Lys Ser 1 5 428 9 PRT homo sapiens 428 Arg Asn Leu Phe Cys Ser Tyr Gly Leu 1 5 429 9 PRT homo sapiens 429 Trp Phe Asp Ser Thr Asp Gly Thr Phe 1 5 430 9 PRT homo sapiens 430 Arg Leu His Glu Ala Ser Glu Asn Leu 1 5 431 9 PRT homo sapiens 431 Asn Ser Gln Pro Leu Asn Leu Ala Leu 1 5 432 9 PRT homo sapiens 432 Val Leu Leu Ile Val Leu Phe Cys Leu 1 5 433 9 PRT homo sapiens 433 Leu Leu Ala Pro Val Phe Arg Thr Leu 1 5 434 9 PRT homo sapiens 434 Trp Tyr Pro Gln Ala Glu Val Gln Asn 1 5 435 9 PRT homo sapiens 435 Ser Pro Gln Gln Ser Ala Gln Leu Leu 1 5 436 9 PRT homo sapiens 436 Leu Gln Leu Thr Leu Thr Ala Phe Leu 1 5 437 9 PRT homo sapiens 437 Leu Val Pro Ser Leu Thr Arg Tyr Leu 1 5 438 9 PRT homo sapiens 438 Thr Ile Leu Val Gln Pro Gln His Leu 1 5 439 9 PRT homo sapiens 439 Ser Tyr Arg Lys Val Thr Trp His Trp 1 5 440 9 PRT homo sapiens 440 Asp Tyr Lys Trp Val Asp Arg Asn Ser 1 5 441 9 PRT homo sapiens 441 Leu Thr Asn Gln Ser Asn Cys Trp Leu 1 5 442 9 PRT homo sapiens 442 Asn Tyr Ser Glu Glu Ile Lys Ser Asn 1 5 443 9 PRT homo sapiens 443 Ile Leu Val Gln Pro Gln His Leu Leu 1 5 444 9 PRT homo sapiens 444 Val Ser Asn Arg Ala Met Lys Gly Leu 1 5 445 9 PRT homo sapiens 445 Leu Ser Asn Cys Ala Leu Leu Gln Leu 1 5 446 9 PRT homo sapiens 446 Val Asn Asn Ser Gly Leu Phe Phe Leu 1 5 447 9 PRT homo sapiens 447 Ser Cys Gln Asn Gln Thr Lys Gly Leu 1 5 448 9 PRT homo sapiens 448 Leu Ser Pro Gln Gln Ser Ala Gln Leu 1 5 449 9 PRT homo sapiens 449 Gly Ser Leu Ser Asn Cys Ala Leu Leu 1 5 450 9 PRT homo sapiens 450 Ser Leu Asn Ser Gln Pro Leu Asn Leu 1 5 451 9 PRT homo sapiens 451 Gly Thr Val Gly Lys Gln Cys Cys Leu 1 5 452 9 PRT homo sapiens 452 Cys Gln Asn Gln Thr Lys Gly Leu Leu 1 5 453 9 PRT homo sapiens 453 Glu Asn Leu Lys Asn Val Pro Leu Leu 1 5 454 9 PRT homo sapiens 454 Thr Trp Asn Ser Ser Ala Val Pro Leu 1 5 455 9 PRT homo sapiens 455 Thr Ile Arg Ala Leu Phe Pro Ser Leu 1 5 456 9 PRT homo sapiens 456 Leu Leu Tyr Gln Leu Phe Arg Asn Leu 1 5 457 9 PRT homo sapiens 457 Val Tyr Lys Gly Phe Pro Pro Lys Trp 1 5 458 9 PRT homo sapiens 458 Leu Tyr Cys Asn Pro Lys Asp Asn Ser 1 5 459 9 PRT homo sapiens 459 Asn Cys Ala Leu Leu Gln Leu Thr Leu 1 5 460 9 PRT homo sapiens 460 Gln Thr Lys Gly Leu Leu Tyr Gln Leu 1 5 461 9 PRT homo sapiens 461 Ser Asn Cys Trp Leu Cys Glu His Leu 1 5 462 9 PRT homo sapiens 462 Glu Ala Ser Met Glu Ala Gln Gly Leu 1 5 463 9 PRT homo sapiens 463 Trp Gly Tyr Val Leu Leu Ile Val Leu 1 5 464 9 PRT homo sapiens 464 Gly Leu Gly Tyr Leu Val Pro Ser Leu 1 5 465 10 PRT homo sapiens 465 Arg Tyr Leu Thr Leu Asn Ala Ser Gln Ile 1 5 10 466 10 PRT homo sapiens 466 Asn Tyr Ser Glu Glu Ile Lys Ser Asn Ile 1 5 10 467 10 PRT homo sapiens 467 Leu Phe Pro Ser Leu Gly Thr Tyr Asp Leu 1 5 10 468 10 PRT homo sapiens 468 Trp Phe Arg Ser Trp Gly Tyr Val Leu Leu 1 5 10 469 10 PRT homo sapiens 469 Ile Phe Val Leu Ile Tyr Val Arg Val Phe 1 5 10 470 10 PRT homo sapiens 470 Arg Ser Leu Asn Ser Gln Pro Leu Asn Leu 1 5 10 471 10 PRT homo sapiens 471 Gly Tyr Val Leu Leu Ile Val Leu Phe Cys 1 5 10 472 10 PRT homo sapiens 472 His Leu Leu Ala Pro Val Phe Arg Thr Leu 1 5 10 473 10 PRT homo sapiens 473 Ser Thr Ile Arg Ala Leu Phe Pro Ser Leu 1 5 10 474 10 PRT homo sapiens 474 Gly Leu Leu Tyr Gln Leu Phe Arg Asn Leu 1 5 10 475 10 PRT homo sapiens 475 Tyr Leu Val Pro Ser Leu Thr Arg Tyr Leu 1 5 10 476 10 PRT homo sapiens 476 Leu Phe Cys Leu Phe Ile Phe Val Leu Ile 1 5 10 477 10 PRT homo sapiens 477 Tyr Val Leu Leu Ile Val Leu Phe Cys Leu 1 5 10 478 10 PRT homo sapiens 478 Arg Val Phe Arg Lys Ser Arg Arg Ser Leu 1 5 10 479 10 PRT homo sapiens 479 Arg Thr Trp Asn Ser Ser Ala Val Pro Leu 1 5 10 480 10 PRT homo sapiens 480 Leu Tyr Gln Leu Phe Arg Asn Leu Phe Cys 1 5 10 481 10 PRT homo sapiens 481 Gly Tyr Leu Val Pro Ser Leu Thr Arg Tyr 1 5 10 482 10 PRT homo sapiens 482 Met Tyr Glu Arg Val Trp Tyr Pro Gln Ala 1 5 10 483 10 PRT homo sapiens 483 Gln Ser Asn Cys Trp Leu Cys Glu His Leu 1 5 10 484 10 PRT homo sapiens 484 Leu Thr Ile Leu Val Gln Pro Gln His Leu 1 5 10 485 10 PRT homo sapiens 485 Leu Ser Pro Gln Gln Ser Ala Gln Leu Leu 1 5 10 486 10 PRT homo sapiens 486 Leu Leu Gln Leu Thr Leu Thr Ala Phe Leu 1 5 10 487 10 PRT homo sapiens 487 Cys Gly Leu Gly Tyr Leu Val Pro Ser Leu 1 5 10 488 10 PRT homo sapiens 488 Gln Gly Leu Ser Phe Ala Gln Val Arg Leu 1 5 10 489 10 PRT homo sapiens 489 Ala Pro Val Phe Arg Thr Leu Ser Ile Leu 1 5 10 490 10 PRT homo sapiens 490 Thr Leu Asn Ala Ser Gln Ile Thr Asn Leu 1 5 10 491 10 PRT homo sapiens 491 Ala Ser Glu Asn Leu Lys Asn Val Pro Leu 1 5 10 492 10 PRT homo sapiens 492 Leu Thr Leu Ser Val Asn Asn Ser Gly Leu 1 5 10 493 10 PRT homo sapiens 493 Ser Cys Gln Asn Gln Thr Lys Gly Leu Leu 1 5 10 494 10 PRT homo sapiens 494 Trp Trp Leu Thr Gly Ser Asn Leu Thr Leu 1 5 10 495 10 PRT homo sapiens 495 Leu Tyr Cys Asn Pro Lys Asp Asn Ser Thr 1 5 10 496 10 PRT homo sapiens 496 Ser Val Asn Asn Ser Gly Leu Phe Phe Leu 1 5 10 497 10 PRT homo sapiens 497 Leu Thr Leu Thr Ala Phe Leu Thr Ile Leu 1 5 10 498 10 PRT homo sapiens 498 Thr Ile Leu Val Gln Pro Gln His Leu Leu 1 5 10 499 10 PRT homo sapiens 499 Thr Tyr Asp Leu Glu Lys Ala Ile Leu Asn 1 5 10 500 10 PRT homo sapiens 500 Val Tyr Lys Gly Phe Pro Pro Lys Trp Ser 1 5 10 501 10 PRT homo sapiens 501 Ser Trp Gly Tyr Val Leu Leu Ile Val Leu 1 5 10 502 10 PRT homo sapiens 502 Asn Gln Thr Lys Gly Leu Leu Tyr Gln Leu 1 5 10 503 10 PRT homo sapiens 503 Gly Thr Tyr Asp Leu Glu Lys Ala Ile Leu 1 5 10 504 10 PRT homo sapiens 504 Leu Asn Ser Gln Pro Leu Asn Leu Ala Leu 1 5 10 505 10 PRT homo sapiens 505 Ile Pro Lys Gln Tyr Cys Asn Gln Ile Leu 1 5 10 506 10 PRT homo sapiens 506 Ser Asn Cys Ala Leu Leu Gln Leu Thr Leu 1 5 10 507 10 PRT homo sapiens 507 Thr Gln Gly Asp Thr Asp Asn Pro Pro Leu 1 5 10 508 10 PRT homo sapiens 508 Ala Leu Ser Pro Gln Gln Ser Ala Gln Leu 1 5 10 509 10 PRT homo sapiens 509 His Leu Asp Asn Ala Glu Gln Pro Glu Leu 1 5 10 510 10 PRT homo sapiens 510 Val Leu Leu Ile Val Leu Phe Cys Leu Phe 1 5 10 511 10 PRT homo sapiens 511 Gln Val Ser Asn Arg Ala Met Lys Gly Leu 1 5 10 512 10 PRT homo sapiens 512 Val Pro Ser Leu Thr Arg Tyr Leu Thr Leu 1 5 10 513 10 PRT homo sapiens 513 Met Gly Ser Leu Ser Asn Cys Ala Leu Leu 1 5 10 514 10 PRT homo sapiens 514 Asp Trp Phe Arg Ser Trp Gly Tyr Val Leu 1 5 10 515 9 PRT homo sapiens 515 Phe Pro Ser Leu Gly Thr Tyr Asp Leu 1 5 516 9 PRT homo sapiens 516 Ser Pro Gln Gln Ser Ala Gln Leu Leu 1 5 517 9 PRT homo sapiens 517 Thr Ile Arg Ala Leu Phe Pro Ser Leu 1 5 518 9 PRT homo sapiens 518 Ser Gly Arg Cys Gly Leu Gly Tyr Leu 1 5 519 9 PRT homo sapiens 519 Ala Pro Val Phe Arg Thr Leu Ser Ile 1 5 520 9 PRT homo sapiens 520 Leu Val Pro Ser Leu Thr Arg Tyr Leu 1 5 521 9 PRT homo sapiens 521 Glu Ala Ser Met Glu Ala Gln Gly Leu 1 5 522 9 PRT homo sapiens 522 Ser Ala Ser Arg Lys Asp His Val Leu 1 5 523 9 PRT homo sapiens 523 Val Pro Leu Leu Asp Trp Gln Gly Ile 1 5 524 9 PRT homo sapiens 524 Ile Pro Lys Gln Tyr Cys Asn Gln Ile 1 5 525 9 PRT homo sapiens 525 Ile Leu Val Gln Pro Gln His Leu Leu 1 5 526 9 PRT homo sapiens 526 Ser Ser Ala Val Pro Leu Ile Gly Leu 1 5 527 9 PRT homo sapiens 527 Gly His Arg Thr Pro Thr Trp Trp Leu 1 5 528 9 PRT homo sapiens 528 Val Phe Arg Lys Ser Arg Arg Ser Leu 1 5 529 9 PRT homo sapiens 529 Gln Thr Lys Gly Leu Leu Tyr Gln Leu 1 5 530 9 PRT homo sapiens 530 Glu Ile Lys Ser Asn Ile Gln Arg Leu 1 5 531 9 PRT homo sapiens 531 Gly Leu Ser Phe Ala Gln Val Arg Leu 1 5 532 9 PRT homo sapiens 532 Val Leu Leu Ile Val Leu Phe Cys Leu 1 5 533 9 PRT homo sapiens 533 Trp Phe Arg Ser Trp Gly Tyr Val Leu 1 5 534 9 PRT homo sapiens 534 Trp Leu Thr Gly Ser Asn Leu Thr Leu 1 5 535 9 PRT homo sapiens 535 Leu Ser Asn Cys Ala Leu Leu Gln Leu 1 5 536 9 PRT homo sapiens 536 Val Ser Asn Arg Ala Met Lys Gly Leu 1 5 537 9 PRT homo sapiens 537 Asn Ser Gln Pro Leu Asn Leu Ala Leu 1 5 538 9 PRT homo sapiens 538 Ser Asn Cys Trp Leu Cys Glu His Leu 1 5 539 9 PRT homo sapiens 539 Ser Cys Gln Asn Gln Thr Lys Gly Leu 1 5 540 9 PRT homo sapiens 540 Leu Leu Tyr Gln Leu Phe Arg Asn Leu 1 5 541 9 PRT homo sapiens 541 Leu Ser Pro Gln Gln Ser Ala Gln Leu 1 5 542 9 PRT homo sapiens 542 Thr Leu Thr Ala Phe Leu Thr Ile Leu 1 5 543 9 PRT homo sapiens 543 Leu Gln Leu Thr Leu Thr Ala Phe Leu 1 5 544 9 PRT homo sapiens 544 Trp Gly Tyr Val Leu Leu Ile Val Leu 1 5 545 9 PRT homo sapiens 545 Leu Leu Ala Pro Val Phe Arg Thr Leu 1 5 546 9 PRT homo sapiens 546 Leu Ser Phe Ala Gln Val Arg Leu Leu 1 5 547 9 PRT homo sapiens 547 Glu Asn Leu Lys Asn Val Pro Leu Leu 1 5 548 9 PRT homo sapiens 548 Ser Leu Asn Ser Gln Pro Leu Asn Leu 1 5 549 9 PRT homo sapiens 549 Gly Ser Leu Ser Asn Cys Ala Leu Leu 1 5 550 9 PRT homo sapiens 550 Thr Ile Leu Val Gln Pro Gln His Leu 1 5 551 9 PRT homo sapiens 551 Asn Cys Ala Leu Leu Gln Leu Thr Leu 1 5 552 9 PRT homo sapiens 552 Gly Leu Gly Tyr Leu Val Pro Ser Leu 1 5 553 9 PRT homo sapiens 553 Leu Asn Ala Ser Gln Ile Thr Asn Leu 1 5 554 9 PRT homo sapiens 554 Arg Leu His Glu Ala Ser Glu Asn Leu 1 5 555 9 PRT homo sapiens 555 Met Gly Ser Leu Ser Asn Cys Ala Leu 1 5 556 9 PRT homo sapiens 556 Thr Leu Ser Val Asn Asn Ser Gly Leu 1 5 557 9 PRT homo sapiens 557 Gln Pro Gln His Leu Leu Ala Pro Val 1 5 558 9 PRT homo sapiens 558 Gly Thr Val Gly Lys Gln Cys Cys Leu 1 5 559 9 PRT homo sapiens 559 Cys Gln Asn Gln Thr Lys Gly Leu Leu 1 5 560 9 PRT homo sapiens 560 Arg Asn Leu Phe Cys Ser Tyr Gly Leu 1 5 561 9 PRT homo sapiens 561 Leu Thr Asn Gln Ser Asn Cys Trp Leu 1 5 562 9 PRT homo sapiens 562 Arg Leu Leu Glu Gly Asn Phe Ser Leu 1 5 563 9 PRT homo sapiens 563 Val Asn Asn Ser Gly Leu Phe Phe Leu 1 5 564 9 PRT homo sapiens 564 Thr Thr His Gln Tyr Asp Thr Ser Leu 1 5 565 10 PRT homo sapiens 565 Ala Pro Val Phe Arg Thr Leu Ser Ile Leu 1 5 10 566 10 PRT homo sapiens 566 Ile Pro Lys Gln Tyr Cys Asn Gln Ile Leu 1 5 10 567 10 PRT homo sapiens 567 Val Pro Ser Leu Thr Arg Tyr Leu Thr Leu 1 5 10 568 10 PRT homo sapiens 568 Arg Val Phe Arg Lys Ser Arg Arg Ser Leu 1 5 10 569 10 PRT homo sapiens 569 Tyr Val Leu Leu Ile Val Leu Phe Cys Leu 1 5 10 570 10 PRT homo sapiens 570 Ser Val Asn Asn Ser Gly Leu Phe Phe Leu 1 5 10 571 10 PRT homo sapiens 571 Gln Val Ser Asn Arg Ala Met Lys Gly Leu 1 5 10 572 10 PRT homo sapiens 572 Pro Pro Lys Trp Ser Gly Arg Cys Gly Leu 1 5 10 573 10 PRT homo sapiens 573 Ala Ser Ala Ser Arg Lys Asp His Val Leu 1 5 10 574 10 PRT homo sapiens 574 Ala Leu Ser Pro Gln Gln Ser Ala Gln Leu 1 5 10 575 10 PRT homo sapiens 575 Glu Ala His Gln Ser Lys Val Ser Ser Leu 1 5 10 576 10 PRT homo sapiens 576 Ala Ser Arg Lys Asp His Val Leu Asp Ile 1 5 10 577 10 PRT homo sapiens 577 Asn Ser Ser Ala Val Pro Leu Ile Gly Leu 1 5 10 578 10 PRT homo sapiens 578 Asp Gly His Arg Thr Pro Thr Trp Trp Leu 1 5 10 579 10 PRT homo sapiens 579 Thr Ile Leu Val Gln Pro Gln His Leu Leu 1 5 10 580 10 PRT homo sapiens 580 Lys Val Thr Trp His Trp Glu Ala Ser Met 1 5 10 581 10 PRT homo sapiens 581 Gln Gly Leu Ser Phe Ala Gln Val Arg Leu 1 5 10 582 10 PRT homo sapiens 582 Leu Thr Trp Ser Gly Asn Asp Thr Cys Leu 1 5 10 583 10 PRT homo sapiens 583 Leu Ser Pro Gln Gln Ser Ala Gln Leu Leu 1 5 10 584 10 PRT homo sapiens 584 Thr Leu Asn Ala Ser Gln Ile Thr Asn Leu 1 5 10 585 10 PRT homo sapiens 585 Ser Thr Ile Arg Ala Leu Phe Pro Ser Leu 1 5 10 586 10 PRT homo sapiens 586 Cys Gly Thr Val Gly Lys Gln Cys Cys Leu 1 5 10 587 10 PRT homo sapiens 587 Gly Leu Ser Phe Ala Gln Val Arg Leu Leu 1 5 10 588 10 PRT homo sapiens 588 Ser Cys Gln Asn Gln Thr Lys Gly Leu Leu 1 5 10 589 10 PRT homo sapiens 589 Thr Gln Gly Asp Thr Asp Asn Pro Pro Leu 1 5 10 590 10 PRT homo sapiens 590 Arg Thr Trp Asn Ser Ser Ala Val Pro Leu 1 5 10 591 10 PRT homo sapiens 591 Arg Ser Leu Asn Ser Gln Pro Leu Asn Leu 1 5 10 592 10 PRT homo sapiens 592 Gln Ser Asn Cys Trp Leu Cys Glu His Leu 1 5 10 593 10 PRT homo sapiens 593 Leu Asn Ser Gln Pro Leu Asn Leu Ala Leu 1 5 10 594 10 PRT homo sapiens 594 Gly Leu Leu Tyr Gln Leu Phe Arg Asn Leu 1 5 10 595 10 PRT homo sapiens 595 His Leu Leu Ala Pro Val Phe Arg Thr Leu 1 5 10 596 10 PRT homo sapiens 596 Asn Gln Thr Lys Gly Leu Leu Tyr Gln Leu 1 5 10 597 10 PRT homo sapiens 597 Leu Pro Asn Thr Gln Asp Tyr Lys Trp Val 1 5 10 598 10 PRT homo sapiens 598 Glu Asn Lys Asn Gly Ser Gly Pro Phe Leu 1 5 10 599 10 PRT homo sapiens 599 Trp Phe Arg Ser Trp Gly Tyr Val Leu Leu 1 5 10 600 10 PRT homo sapiens 600 Leu Thr Leu Thr Ala Phe Leu Thr Ile Leu 1 5 10 601 10 PRT homo sapiens 601 Ser Pro Gln Gln Ser Ala Gln Leu Leu Val 1 5 10 602 10 PRT homo sapiens 602 Leu Thr Thr His Gln Tyr Asp Thr Ser Leu 1 5 10 603 10 PRT homo sapiens 603 Cys Gly Leu Gly Tyr Leu Val Pro Ser Leu 1 5 10 604 10 PRT homo sapiens 604 Leu Thr Leu Ser Val Asn Asn Ser Gly Leu 1 5 10 605 10 PRT homo sapiens 605 Gly Thr Tyr Asp Leu Glu Lys Ala Ile Leu 1 5 10 606 10 PRT homo sapiens 606 Leu Thr Ile Leu Val Gln Pro Gln His Leu 1 5 10 607 10 PRT homo sapiens 607 Thr Thr His Gln Tyr Asp Thr Ser Leu Leu 1 5 10 608 10 PRT homo sapiens 608 Ile Leu Thr Asn Gln Ser Asn Cys Trp Leu 1 5 10 609 10 PRT homo sapiens 609 Ser Arg Arg Ser Leu Asn Ser Gln Pro Leu 1 5 10 610 10 PRT homo sapiens 610 Leu Leu Gln Leu Thr Leu Thr Ala Phe Leu 1 5 10 611 10 PRT homo sapiens 611 Tyr Leu Val Pro Ser Leu Thr Arg Tyr Leu 1 5 10 612 10 PRT homo sapiens 612 Val Leu Phe Cys Leu Phe Ile Phe Val Leu 1 5 10 613 10 PRT homo sapiens 613 Ser Asn Cys Ala Leu Leu Gln Leu Thr Leu 1 5 10 614 10 PRT homo sapiens 614 Ser Leu Ser Asn Cys Ala Leu Leu Gln Leu 1 5 10 615 9 PRT homo sapiens 615 Ile Pro Lys Gln Tyr Cys Asn Gln Ile 1 5 616 9 PRT homo sapiens 616 Phe Pro Ser Leu Gly Thr Tyr Asp Leu 1 5 617 9 PRT homo sapiens 617 Ser Pro Gln Gln Ser Ala Gln Leu Leu 1 5 618 9 PRT homo sapiens 618 Trp Ser Gly Asn Asp Thr Cys Leu Tyr 1 5 619 9 PRT homo sapiens 619 Ile Ser Lys Ala Met Glu Gln Glu Phe 1 5 620 9 PRT homo sapiens 620 Val Pro Leu Leu Asp Trp Gln Gly Ile 1 5 621 9 PRT homo sapiens 621 Ala Ser Ala Ser Thr Trp Trp Thr Tyr 1 5 622 9 PRT homo sapiens 622 Trp Ser Gly Arg Cys Gly Leu Gly Tyr 1 5 623 9 PRT homo sapiens 623 Val Pro Ala Ser Ala Ser Thr Trp Trp 1 5 624 9 PRT homo sapiens 624 Leu Pro Asn Thr Gln Asp Tyr Lys Trp 1 5 625 9 PRT homo sapiens 625 Ala Pro Val Phe Arg Thr Leu Ser Ile 1 5 626 9 PRT homo sapiens 626 Leu Ser Pro Gln Gln Ser Ala Gln Leu 1 5 627 9 PRT homo sapiens 627 Asn Ser Gln Pro Leu Asn Leu Ala Leu 1 5 628 9 PRT homo sapiens 628 Val Ser Asn Arg Ala Met Lys Gly Leu 1 5 629 9 PRT homo sapiens 629 Ser Ser Ala Val Pro Leu Ile Gly Leu 1 5 630 9 PRT homo sapiens 630 Leu Ser Phe Ala Gln Val Arg Leu Leu 1 5 631 9 PRT homo sapiens 631 Gly Ser Leu Ser Asn Cys Ala Leu Leu 1 5 632 9 PRT homo sapiens 632 Leu Ser Val Asn Asn Ser Gly Leu Phe 1 5 633 9 PRT homo sapiens 633 Leu Ser Asn Cys Ala Leu Leu Gln Leu 1 5 634 9 PRT homo sapiens 634 Glu Ala Ser Met Glu Ala Gln Gly Leu 1 5 635 9 PRT homo sapiens 635 Gln Pro Gln His Leu Leu Ala Pro Val 1 5 636 9 PRT homo sapiens 636 Arg Leu Leu Glu Gly Asn Phe Ser Leu 1 5 637 9 PRT homo sapiens 637 Arg Leu His Glu Ala Ser Glu Asn Leu 1 5 638 9 PRT homo sapiens 638 Lys Gln Cys Cys Leu Tyr Ile Asn Tyr 1 5 639 9 PRT homo sapiens 639 Arg Ser Trp Gly Tyr Val Leu Leu Ile 1 5 640 9 PRT homo sapiens 640 Ser Gly Arg Cys Gly Leu Gly Tyr Leu 1 5 641 9 PRT homo sapiens 641 Gln Thr Lys Gly Leu Leu Tyr Gln Leu 1 5 642 9 PRT homo sapiens 642 Glu Ile Lys Ser Asn Ile Gln Arg Leu 1 5 643 9 PRT homo sapiens 643 Ser Ala Ser Arg Lys Asp His Val Leu 1 5 644 9 PRT homo sapiens 644 Glu Asn Lys Asn Gly Ser Gly Pro Phe 1 5 645 9 PRT homo sapiens 645 Thr Ile Arg Ala Leu Phe Pro Ser Leu 1 5 646 9 PRT homo sapiens 646 Gln Val Arg Leu Leu Glu Gly Asn Phe 1 5 647 9 PRT homo sapiens 647 Thr Ser Ser Tyr Arg Lys Val Thr Trp 1 5 648 9 PRT homo sapiens 648 Glu Ser Arg Asn Asp Asp Asp Asp Thr 1 5 649 9 PRT homo sapiens 649 Trp Thr Tyr Ser Gly Gln Trp Met Tyr 1 5 650 9 PRT homo sapiens 650 Lys Val Gly Asp Trp Phe Arg Ser Trp 1 5 651 9 PRT homo sapiens 651 Val Pro Leu Ile Gly Leu Pro Asn Thr 1 5 652 9 PRT homo sapiens 652 Phe Pro Pro Lys Trp Ser Gly Arg Cys 1 5 653 9 PRT homo sapiens 653 Val Pro Ser Leu Thr Arg Tyr Leu Thr 1 5 654 9 PRT homo sapiens 654 Ile Gly Leu Pro Asn Thr Gln Asp Tyr 1 5 655 9 PRT homo sapiens 655 Phe Leu Gly Asn Ile Pro Lys Gln Tyr 1 5 656 9 PRT homo sapiens 656 Cys Leu Phe Ile Phe Val Leu Ile Tyr 1 5 657 9 PRT homo sapiens 657 Ala Ile Leu Asn Ile Ser Lys Ala Met 1 5 658 9 PRT homo sapiens 658 Arg Asn Leu Phe Cys Ser Tyr Gly Leu 1 5 659 9 PRT homo sapiens 659 Thr Val Gly Lys Gln Cys Cys Leu Tyr 1 5 660 9 PRT homo sapiens 660 Tyr Leu Val Pro Ser Leu Thr Arg Tyr 1 5 661 9 PRT homo sapiens 661 Ile Pro Thr Thr Gln Arg Gln Thr Ala 1 5 662 9 PRT homo sapiens 662 Gly Ser Gly Pro Phe Leu Gly Asn Ile 1 5 663 9 PRT homo sapiens 663 Val Thr Trp His Trp Glu Ala Ser Met 1 5 664 9 PRT homo sapiens 664 Thr Pro Thr Trp Trp Leu Thr Gly Ser 1 5 665 10 PRT homo sapiens 665 Ile Pro Lys Gln Tyr Cys Asn Gln Ile Leu 1 5 10 666 10 PRT homo sapiens 666 Val Pro Ser Leu Thr Arg Tyr Leu Thr Leu 1 5 10 667 10 PRT homo sapiens 667 Gln Pro Gln His Leu Leu Ala Pro Val Phe 1 5 10 668 10 PRT homo sapiens 668 Val Pro Leu Leu Asp Trp Gln Gly Ile Phe 1 5 10 669 10 PRT homo sapiens 669 Ala Pro Val Phe Arg Thr Leu Ser Ile Leu 1 5 10 670 10 PRT homo sapiens 670 Asn Pro Lys Asp Asn Ser Thr Ile Arg Ala 1 5 10 671 10 PRT homo sapiens 671 Lys Ala Ile Leu Asn Ile Ser Lys Ala Met 1 5 10 672 10 PRT homo sapiens 672 Arg Ala Leu Phe Pro Ser Leu Gly Thr Tyr 1 5 10 673 10 PRT homo sapiens 673 Arg Ser Leu Asn Ser Gln Pro Leu Asn Leu 1 5 10 674 10 PRT homo sapiens 674 Ala Ser Met Glu Ala Gln Gly Leu Ser Phe 1 5 10 675 10 PRT homo sapiens 675 Thr Ser Cys Gln Val Ser Asn Arg Ala Met 1 5 10 676 10 PRT homo sapiens 676 Ala Met Lys Gly Leu Thr Thr His Gln Tyr 1 5 10 677 10 PRT homo sapiens 677 Pro Pro Lys Trp Ser Gly Arg Cys Gly Leu 1 5 10 678 10 PRT homo sapiens 678 Leu Pro Asn Thr Gln Asp Tyr Lys Trp Val 1 5 10 679 10 PRT homo sapiens 679 Ala Ser Arg Lys Asp His Val Leu Asp Ile 1 5 10 680 10 PRT homo sapiens 680 Leu Ser Pro Gln Gln Ser Ala Gln Leu Leu 1 5 10 681 10 PRT homo sapiens 681 Tyr Ser Cys Gln Asn Gln Thr Lys Gly Leu 1 5 10 682 10 PRT homo sapiens 682 Ala Ser Gln Ile Thr Asn Leu Arg Ser Phe 1 5 10 683 10 PRT homo sapiens 683 Trp Ser Gly Arg Cys Gly Leu Gly Tyr Leu 1 5 10 684 10 PRT homo sapiens 684 Gln Ser Asn Cys Trp Leu Cys Glu His Leu 1 5 10 685 10 PRT homo sapiens 685 Ala Ser Ala Ser Arg Lys Asp His Val Leu 1 5 10 686 10 PRT homo sapiens 686 Asn Ser Ser Ala Val Pro Leu Ile Gly Leu 1 5 10 687 10 PRT homo sapiens 687 Leu Ser Val Asn Asn Ser Gly Leu Phe Phe 1 5 10 688 10 PRT homo sapiens 688 Ser Pro Gln Gln Ser Ala Gln Leu Leu Val 1 5 10 689 10 PRT homo sapiens 689 Lys Val Thr Trp His Trp Glu Ala Ser Met 1 5 10 690 10 PRT homo sapiens 690 Thr Gln Gly Asp Thr Asp Asn Pro Pro Leu 1 5 10 691 10 PRT homo sapiens 691 Gln Thr Lys Gly Leu Leu Tyr Gln Leu Phe 1 5 10 692 10 PRT homo sapiens 692 Glu Asn Lys Asn Gly Ser Gly Pro Phe Leu 1 5 10 693 10 PRT homo sapiens 693 Gly Thr Tyr Asp Leu Glu Lys Ala Ile Leu 1 5 10 694 10 PRT homo sapiens 694 Glu Ala His Gln Ser Lys Val Ser Ser Leu 1 5 10 695 10 PRT homo sapiens 695 Gly Gln Trp Met Tyr Glu Arg Val Trp Tyr 1 5 10 696 10 PRT homo sapiens 696 Ser Ser Tyr Arg Lys Val Thr Trp His Trp 1 5 10 697 10 PRT homo sapiens 697 Ile Ser Lys Ala Met Glu Gln Glu Phe Ser 1 5 10 698 10 PRT homo sapiens 698 Glu Ser Arg Asn Asp Asp Asp Asp Thr Ser 1 5 10 699 10 PRT homo sapiens 699 Gly Thr Val Gly Lys Gln Cys Cys Leu Tyr 1 5 10 700 10 PRT homo sapiens 700 Tyr Pro Gln Ala Glu Val Gln Asn His Ser 1 5 10 701 10 PRT homo sapiens 701 Cys Gln Asn Gln Thr Lys Gly Leu Leu Tyr 1 5 10 702 10 PRT homo sapiens 702 Gln Leu Phe Arg Asn Leu Phe Cys Ser Tyr 1 5 10 703 10 PRT homo sapiens 703 Val Pro Ala Ser Ala Ser Thr Trp Trp Thr 1 5 10 704 10 PRT homo sapiens 704 Arg Val Phe Arg Lys Ser Arg Arg Ser Leu 1 5 10 705 10 PRT homo sapiens 705 Asp Asn Ala Glu Gln Pro Glu Leu Val Phe 1 5 10 706 10 PRT homo sapiens 706 Met Pro Ser Ile Asp Val Thr Asn Glu Ser 1 5 10 707 10 PRT homo sapiens 707 Thr Pro His Arg Cys Thr Gln Gly Asp Thr 1 5 10 708 10 PRT homo sapiens 708 Arg Ser Trp Gly Tyr Val Leu Leu Ile Val 1 5 10 709 10 PRT homo sapiens 709 Thr Pro Thr Trp Trp Leu Thr Gly Ser Asn 1 5 10 710 10 PRT homo sapiens 710 Lys Gln Tyr Cys Asn Gln Ile Leu Trp Phe 1 5 10 711 10 PRT homo sapiens 711 Arg Thr Trp Asn Ser Ser Ala Val Pro Leu 1 5 10 712 10 PRT homo sapiens 712 Glu Val Gln Asn His Ser Thr Ser Ser Tyr 1 5 10 713 10 PRT homo sapiens 713 Leu Ile Gly Leu Pro Asn Thr Gln Asp Tyr 1 5 10 714 10 PRT homo sapiens 714 Phe Cys Leu Phe Ile Phe Val Leu Ile Tyr 1 5 10 715 4 PRT homo sapiens 715 Asn Gln Ser Asn 1 716 4 PRT homo sapiens 716 Asn His Ser Thr 1 717 4 PRT homo sapiens 717 Asn Phe Ser Leu 1 718 4 PRT homo sapiens 718 Asn Gly Ser Gly 1 719 4 PRT homo sapiens 719 Asn Glu Ser Arg 1 720 4 PRT homo sapiens 720 Asn Arg Thr Trp 1 721 4 PRT homo sapiens 721 Asn Ser Ser Ala 1 722 4 PRT homo sapiens 722 Asn Asp Thr Cys 1 723 4 PRT homo sapiens 723 Asn Gln Thr Lys 1 724 4 PRT homo sapiens 724 Asn Leu Thr Leu 1 725 4 PRT homo sapiens 725 Asn Asn Ser Gly 1 726 4 PRT homo sapiens 726 Asn Ala Ser Gln 1 727 4 PRT homo sapiens 727 Asn Ser Thr Ile 1 728 4 PRT homo sapiens 728 Asn Ile Ser Lys 1 729 4 PRT homo sapiens 729 Asn Tyr Ser Glu 1 730 4 PRT homo sapiens 730 Arg Lys Val Thr 1 731 4 PRT homo sapiens 731 Ser Arg Asn Asp 1 732 4 PRT homo sapiens 732 Ser Gly Asn Asp 1 733 4 PRT homo sapiens 733 Thr Gln Gly Asp 1 734 4 PRT homo sapiens 734 Ser Arg Lys Asp 1 735 6 PRT homo sapiens 735 Gly Ser Leu Ser Asn Cys 1 5 736 6 PRT homo sapiens 736 Gly Leu Ser Phe Ala Gln 1 5 737 6 PRT homo sapiens 737 Gly Thr Arg Gln Cys Ser 1 5 738 6 PRT homo sapiens 738 Gly Leu Pro Asn Thr Gln 1 5 739 6 PRT homo sapiens 739 Gly Leu Thr Trp Ser Gly 1 5 740 6 PRT homo sapiens 740 Gly Asn Asp Thr Cys Leu 1 5 741 6 PRT homo sapiens 741 Gly Leu Thr Glu Ala His 1 5 742 6 PRT homo sapiens 742 Gly Ser Asn Leu Thr Leu 1 5 743 6 PRT homo sapiens 743 Gly Val Tyr Lys Gly Phe 1 5 744 22 PRT homo sapiens 744 Leu Thr Ala Phe Leu Thr Ile Leu Val Gln Pro Gln His Leu Leu Ala 1 5 10 15 Pro Val Phe Arg Thr Leu 20 745 34 PRT homo sapiens 745 Gly Asp Trp Phe Arg Ser Trp Gly Tyr Val Leu Leu Ile Val Leu Phe 1 5 10 15 Cys Leu Phe Ile Phe Val Leu Ile Tyr Val Arg Val Phe Arg Lys Ser 20 25 30 Arg Arg 746 21 PRT homo sapiens 746 Phe Arg Ser Trp Gly Tyr Val Leu Leu Ile Val Leu Phe Cys Leu Phe 1 5 10 15 Ile Phe Val Leu Ile 20 747 17 PRT homo sapiens 747 Val Leu Leu Ile Val Leu Phe Cys Leu Phe Ile Phe Val Leu Ile Tyr 1 5 10 15 Val 748 24 PRT homo sapiens 748 Met Gly Ser Leu Ser Asn Cys Ala Leu Leu Gln Leu Thr Leu Thr Ala 1 5 10 15 Phe Leu Thr Ile Leu Val Gln Pro 20 749 25 DNA homo sapiens 749 atatcgccgc gctcgtcgtc gacaa 25 750 26 DNA homo sapiens 750 agccacacgc agctcattgt agaagg 26 751 24 DNA homo sapiens 751 cttgggaggt cctagtgcta agtg 24 752 24 DNA homo sapiens 752 caatgaaggg actaacaacc catc 24 

1. A polynucleotide that encodes an 103P2D6-related protein, wherein the polynucleotide is selected from the group consisting of: (a) an isolated polynucleotide comprising the sequence as shown in FIG. 2 (SEQ ID NO: 1), wherein T can also be U; (b) a polynucleotide consisting of the sequence as shown in FIG. 2 (SEQ ID NO: 1), from nucleotide residue number 805 through nucleotide residue number 2493, wherein T can also be U; (c) a polynucleotide that encodes a 103P2D6-related protein whose sequence is encoded by a cDNA contained in the plasmid designated p103P2D6-B or p 103P2D6-2 deposited with American Type Culture Collection as Accession No. PTA-1895 and PTA-1155, respectively; (d) a polynucleotide that encodes an 103P2D6-related protein that is at least 90% identical to the entire amino acid sequence shown in FIG. 2 (SEQ ID NO: 2); and (e) a polynucleotide that is fully complementary to a polynucleotide of any one of (a)-(d).
 2. A polynucleotide of claim 1 That encodes the polypeptide sequence shown in SEQ ID NO:
 2. 3. An isolated fragment of a polynucleotide of claim 1 comprising: (a) at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2, from nucleotide residue number 1 through nucleotide residue number 804; or, (b) at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2, from nucleotide residue number 977 through nucleotide residue number 1036; or, (c) at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2, from nucleotide residue number 1414 through nucleotide residue number 1815; or (d) a polynucleotide whose starting base is in the range of 1-804 of FIG. 2 and whose ending base is in the range of 805-2493 of FIG. 2; or (e) a polynucleotide whose starting base is in the range of 977-1036 of FIG. 2 and whose ending base is in the range of 1037-2493 of FIG. 2; or (f) a polynucleotide whose starting base is in the range of 1414-1815 of FIG. 2 and whose ending base is in the range of 1816-2493 of FIG. 2; or (g) a polynucleotide whose starting base is in the range of 805-976 of FIG. 2 and whose ending base is in the range of 977-2493 of FIG. 2; or (h) a polynucleotide whose starting base is in the range of 805-2493 of FIG. 2 and whose ending base is in the range of 2494-4727 of FIG. 2; or (i) a polynucleotide of (d-g) that is at least 10 nucleotide bases in length; or (j) a polynucleotide that selectively hybridizes under stringent conditions to a polynucleotide of (a)-(g); wherein a range is understood to specifically disclose all whole unit positions thereof and wherein T can be U.
 4. A polynucleotide that encodes an 103P2D6-related protein, wherein the polypeptide includes a motif-bearing amino acid sequence set forth in Table XIX.
 5. A polynucleotide that encodes an 103P2D6-related protein, wherein the polypeptide comprises an HLA class I A1, A2, A3, A24, B7, B27, B58, B62 supermotif, or an HLA class II DR supermotif set forth in Table IV (B) or an Alexander pan DR binding epitope supermotif or an HLA DR3 motif.
 6. A polynucleotide of any one of claims 1-4 that is labeled with a detectable marker.
 7. A recombinant expression vector that contains a polynucleotide of any one of claims 1-4.
 8. A host cell that contains an expression vector of claim
 7. 9. A process for producing a 103P2D6-related protein comprising culturing a host cell of claim 8 under conditions sufficient for the production of the polypeptide and recovering the 103P2D6-related protein so produced.
 10. A 103P2D6-related protein produced by the process of claim
 9. 11. An isolated 103P2D6-related protein.
 12. The 103P2D6-related protein of claim 11, wherein 103P2D6-related protein has the amino acid sequence shown in SEQ ID NO:
 2. 13. An isolated 103P2D6-related protein of claim 11 that has an amino acid sequence which is exactly that of an amino acid sequence encoded by a polynucleotide selected from the group consisting of: (a) a polynucleotide consisting of the sequence as shown in SEQ ID NO: 1, wherein T can also be U; (b) a polynucleotide consisting of the sequence as shown in SEQ ID NO: 1, from nucleotide residue number 805 through nucleotide residue number 2493, wherein T can also be U; (c) a polynucleotide that encodes a 103P2D6-related protein whose sequence is encoded by a cDNA contained in the plasmid designated p103P2D6-B or p103P2D6-2 deposited with American Type Culture Collection as Accession No. PTA-1895 and PTA-1155, respectively; (d) a polynucleotide that encodes an 103P2D6-related protein that is at least 90% identical to the entire amino acid sequence shown in SEQ ID NO: 2; and (e) a polynucleotide that is fully complementary to a polynucleotide of any one of (a)-(d).
 14. An isolated 103P2D6-related protein of claim 13 encoded by a polynucleotide selected from the group consisting of: (a) of at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2 (SEQ ID NO: 1), from nucleotide residue number 1 through nucleotide residue number 255; or (b) of at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2 (SEQ ID NO: 1), from nucleotide residue number 730 through nucleotide residue number 997; or (c) a polynucleotide whose starting base is in the range of 1-255 of FIG. 2 (SEQ ID NO: 1) and whose ending base is in the range of 256-2198 of FIG. 2 (SEQ ID NO: 1); or (d) a polynucleotide whose starting base is in the range of 1-729 of FIG. 2 (SEQ ID NO: 1) and whose ending base is in the range of 730-2198 of FIG. 2 (SEQ ID NO: 1); or (e) a polynucleotide whose starting base is in the range of 1-255 of FIG. 2 (SEQ ID NO: 1) and whose ending base is in the range of 175-1773 of FIG. 2 (SEQ ID NO: 1); or (f) a polynucleotide whose starting base is in the range of 730-997 of FIG. 2 (SEQ ID NO: 1) and whose ending base is in the range of 739-1773 of FIG. 2 (SEQ ID NO: 1); or (g) a nucleotide that starts at any of the following positions and ends at a higher position of FIG. 2 (SEQ ID NO 1): 1, 255, a range of 1-255, a range of 256-729; 730, a range of 730-997, 997, 998-1771, a range of 1771-1947, 1947, 1948, a range of 1948-2198, 2198; (h) a polynucleotide of (c-g) that is at least 10 nucleotide bases in length; or (i) a polynucleotide that selectively hybridizes under stringent conditions to a polynucleotide of (a)-(h); wherein a range is understood to specifically disclose all whole unit positions thereof and wherein T can be U.
 15. An antibody or fragment thereof that specifically binds to a 103P2D6-related protein.
 16. The antibody or fragment thereof of claim 15, which is monoclonal.
 17. A recombinant protein comprising the antigen-binding region of a monoclonal antibody of claim
 16. 18. The antibody or fragment thereof of claim 16, which is labeled with a detectable marker.
 19. The recombinant protein of claim 17, which is labeled with a detectable marker.
 20. The antibody fragment of claim 15,lwhich is an Fab, F(ab′)2, Fv or Sfv fragment.
 21. The antibody of claim 15, which is a human antibody.
 22. The recombinant protein of claim 19, which comprises murine antigen binding region residues and human constant region residues.
 23. A non-human transgenic animal that produces an antibody of claim
 15. 24. A hybridoma that produces an antibody of claim
 15. 25. A single chain monoclonal antibody that comprises the variable domains of the heavy and light chains of a monoclonal antibody of claim
 16. 26. A vector comprising a polynucleotide encoding a single chain monoclonal antibody of claim 25 that immunospecifically binds to a 103P2D6-related protein.
 27. An assay for detecting the presence of a 103P2D6-related protein or polynucleotide in a biological sample comprising: contacting the sample with an antibody or polynucleotide, respectively, that specifically binds to the 103P2D6-related protein or polynucleotide, respectively, and detecting the binding of 103P2D6-related protein or polynucleotide, respectively, in the sample thereto.
 28. An assay of claim 27 or detecting the presence of an 103P2D6-related protein or polynucleotide comprising the steps of: obtaining a sample, evaluating said sample in the presence of an 103P2D6-related protein or polynucleotide, whereby said evaluating step produces a result that indicates the presence or amount of 103P2D6-related protein or polynucleotide, respectively.
 29. An assay of claim 28 for detecting the presence of an 103P2D6 polynucleotide in a biological sample, comprising: (a) contacting the sample with a polynucleotide probe that specifically hybridizes to a polynucleotide encoding an 103P2D6-related protein having an amino acid sequence shown in FIG. 2; and (b) detecting the presence of a hybridization complex formed by the hybridization of the probe with 103P2D6 polynucleotide in the sample, wherein the presence of the hybridization complex indicates the presence of 103P2D6 polynucleotide within the sample.
 30. An assay for detecting the presence of 103P2D6 mRNA in a biological sample comprising: (a) producing cDNA from the sample by reverse transcription using at least one primer; (b) amplifying the cDNA so produced using 103P2D6 polynucleotides as sense and antisense primers to amplify 103P2D6 cDNAs therein; (c) detecting the presence of the amplified 103P2D6 cDNA, wherein the 103P2D6 polynucleotides used as the sense and antisense probes are capable of amplifying the 103P2D6 cDNA contained within the plasmid as deposited with American Type Culture Collection as Accession No. PTA-1895 or PTA-1155.
 31. A method of claim 30, for monitoring 103P2D6 gene products comprising: determining the status of 103P2D6 gene products expressed by cells in a tissue sample from an individual; comparing the status so determined to the status of 103P2D6 gene products in a corresponding normal sample; and identifying the presence of aberrant 103P2D6 gene products in the sample relative to the normal sample.
 32. The method of claim 31, wherein the 103P2D6 gene products are monitored by comparing the polynucleotide sequences of 103P2D6 gene products in the test tissue sample with the polynucleotide sequences of 103P2D6 gene products in a corresponding normal sample.
 33. The method of claim 31, wherein the 103P2D6 gene products are monitored by comparing the levels 103P2D6 gene products in the test tissue sample with the levels of 103P2D6 gene products in the corresponding normal sample.
 34. A method of diagnosing the presence of cancer in an individual comprising: performing the method of claim 32 or 33 whereby the presence of elevated 103P2D6 mRNA or protein expression in the test sample relative to the normal tissue sample provides an indication of the presence of cancer.
 35. The method of claim 34, wherein the cancer occurs in a tissue set forth in Table I.
 36. Use of an 103P2D6-related protein, a vector comprising a polynucleotide encoding a single chain monoclonal antibody that immunospecifically binds to an 103P2D6-related protein, an antisense polynucleotide complementary to a polynucleotide having 103P2D6 coding sequences, or a ribozyme capable of cleaving a polynucleotide having 103P2D6 coding sequences, for the preparation of a composition for treating a patient with a cancer that expresses 103P2D6.
 37. The use of claim 36, wherein the cancer occurs in a tissue set forth in Table I.
 38. A pharmaceutical composition comprising an 103P2D6-related protein, an antibody or fragment thereof that specifically binds to an 103P2D6-related protein, a vector comprising a polynucleotide encoding a single chain monoclonal antibody that immunospecifically binds to an 103P2D6-related protein, a polynucleotide comprising an 103P2D6-related protein coding sequence, an antisense polynucleotide complementary to a polynucleotide having an 103P2D6 coding sequences or a ribozyme capable of cleaving a polynucleotide having 103P2D6 coding sequences and, optionally, a physiologically acceptable carrier.
 39. A method of treating a patient with a cancer that expresses 103P2D6 which comprises administering to said patient a composition of claim 38 comprising a vector that comprises a polynucleotide encoding a single chain monoclonal antibody that immunospecifically binds to an 103P2D6-related protein, such that the vector delivers the single chain monoclonal antibody coding sequence to the cancer cells and the encoded single chain antibody is expressed intracellularly therein.
 40. A method of inhibiting the development of a cancer expressing 103P2D6 in a patient, comprising administering to the patient an effective amount of the vaccine composition of claim
 38. 41. A method of generating an immune response in a mammal comprising exposing the mammal's immune system to an immunogenic portion of an 103P2D6-related protein of claim 38, so that an immune response is generated to 103P2D6.
 42. A method of delivering a cytotoxic agent to a cell that expresses 103P2D6 comprising conjugating the cytotoxic agent to an antibody or fragment thereof of claim 15 that specifically binds to a 103P2D6 epitope and exposing the cell to the antibody-agent conjugate.
 43. A method of inducing an immune response to an 103P2D6 protein, said method comprising: providing a 103P2D6-related protein T cell or B cell epitope; contacting the epitope with an immune system T cell or B cell respectively, whereby the immune system T cell or B cell is induced.
 44. The method of claim 43, wherein the immune system cell is a B cell, whereby the induced B cell generates antibodies that specifically bind to the 103P2D6-related protein.
 45. The method of claim 43, wherein the immune system cell is a T cell that is a cytotoxic T cell (CTL), whereby the activated CTL kills an autologous cell that expresses the 103P2D6 protein.
 46. The method of claim 43, wherein the immune system cell is a T cell that is a helper T cell (HTL), whereby the activated HTL secretes cytokines that facilitate the cytotoxic activity of a CTL or the antibody producing activity of a B cell. 